Methods and kits for detection of coenzyme Q10

ABSTRACT

The invention provides methods for rapid and quantitative extraction and detection of coenzyme Q10 in a sample readily adaptable to high throughput screening methods. The invention further provides reagents and kits for practicing the methods of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/164,328, filed on May 25, 2016; now U.S. Pat. No. 10,114,013, whichis a continuation of U.S. application Ser. No. 13/783,205, filed on Mar.1, 2013, now U.S. Pat. No. 9,360,454, issued on Jun. 7, 2016; whichclaims priority to U.S. Provisional Application No. 61/606,019, filed onMar. 2, 2012. The entire contents of each of the foregoing applicationsare hereby incorporated herein by reference.

BACKGROUND

Coenzyme Q10, also referred to herein as CoQ10, ubiquinone, orubidecarenone, is a popular nutritional supplement and can be found incapsule form in nutritional stores, health food stores, pharmacies, andthe like, as a vitamin-like supplement to help protect the immune systemthrough the antioxidant properties of ubiquinol, the reduced form ofCoQ10. CoQ10 is found throughout most tissues of the human body and thetissues of other mammals. CoQ10 is very lipophilic and, for the mostpart, insoluble in water. The insolubility is related to the 50-carbonatom isoprenoid side chain, of hydrocarbon nature as shown in thefollowing structure of CoQ10.

Coenzyme Q₁₀ (CoQ₁₀) is an integral component of the oxidativephosphorylation machinery in mitochondria and is implicated inhomeostasis of biological membranes and cellular redox status. Thereduced form of CoQ₁₀ (CoQ₁₀H₂) is involved in many key physiochemicalfunctions, including serving as an antioxidant, production of ATP, andintercalation with membrane lipids to maintain structure.

SUMMARY OF THE INVENTION

Studies suggest that the lower percentage of CoQ₁₀H₂ in total CoQ₁₀(T-CoQ10) is associated with mitochondrial cytopathies, diabetes, heartdisease, Parkinson's disease and cancer. Recent studies also suggestthat reduced CoQ₁₀ levels are associated with an increased risk forprostate cancer in patients taking statins and an increased risk ofmetastasis in melanoma patients. This underscores the importance ofdeveloping methods for robust, accurate, sensitive and specificmeasurement of CoQ₁₀ and CoQ₁₀H₂. The invention provides methods forrapid and quantitative extraction and detection of coenzyme Q10 in asample readily adaptable to high throughput screening methods. Theinvention further provides reagents and kits for practicing the methodsof the invention

In one aspect, the invention provides methods for determining the amountof coenzyme Q10 (CoQ10) in a sample by adding a first extraction bufferand a second extraction buffer to the sample that results in phaseseparation of the sample; and spectroscopically analyzing the secondextraction layer to determine the amount of CoQ10.

In another aspect, the invention further provides methods fordetermining the amount of CoQ10 in a sample by adding a first extractionbuffer and a second extraction buffer to the sample; mixing the sample;and analyzing the second extraction layer to determine the amount ofCoQ10, wherein a single extraction with the first extraction buffer andthe second extraction buffer results in detecting an at least 2-foldgreater amount of CoQ10 than using methanol-only extraction.

In another aspect, the invention provides methods for determining theamount of CoQ10 in a sample by adding a first extraction buffer to thesample; heating and mixing the sample; adding a second extraction bufferto the sample that results in phase separation of the sample; heatingand mixing the sample; cooling the sample to ambient temperature; andanalyzing the second extraction buffer layer to determine the amount ofCoQ10 in the sample.

In yet another aspect, the invention provides methods for determiningthe amount of CoQ10 in a sample by adding a first extraction buffer tothe sample; heating and mixing the sample; adding a second extractionbuffer to the sample that results in phase separation of the sample;heating and mixing the sample; cooling the sample to ambienttemperature; and by performing spectroscopic analysis the secondextraction buffer layer to determine the amount of CoQ10 in the sample,wherein a single extraction with the extraction buffer results inextracting at least 2-fold greater amount of CoQ10 than usingmethanol-only extraction followed by Liquid chromatography/massspectrometry/mass spectrometry (LC/MS/MS).

In certain embodiments of the invention, the amount of CoQ10 detected inthe sample is determined spectroscopically by spectroscopic analysis. Incertain embodiments of the invention, the amount of CoQ10 is detectedusing LC/MS/MS. In certain embodiments of the invention, the amount ofCoQ10 extracted using a first extraction buffer and a second extractionbuffer is determined spectroscopically, and the amount of CoQ10 detectedusing methanol-only extraction is determined using LC/MS/MS.

In certain embodiments of the invention, the method is part of a highthroughput screening analysis method. In certain embodiments of theinvention, the entire method is carried out by automation. In certainembodiments of the invention, the entire method is carried out in ashort amount of time. For example, the method is performed usingspectroscopic analysis and the amount of CoQ10 in the sample is detectedin about half of the time or less, about a quarter of the time or less,or about a tenth of the time or less than would be required to performthe analysis using LC/MS/MS. In certain embodiments, the amount of timeto perform analysis on a group of samples using spectroscopic detectionmethods is compared to the amount of time to perform analysis on a groupof samples using LC/MS/MS. In certain embodiments, the amount of time toperform analysis on a group of samples using spectroscopic detectionmethods is less than 5 hours, less than 4 hours, less than 3 hours, lessthan 2 hours, less than 30 minutes, less than 15 minutes, less than 10minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes,less than 2 minutes, less than 1 minutes or less than 30 seconds.

In certain embodiments of the invention, the total amount of CoQ10detected in the sample, wherein the sample is extracted using a firstextraction buffer and a second extraction buffer and the amount of CoQ10is determined spectroscopically, is at least 2-fold greater than thetotal amount of CoQ10 detected in a replicate sample using methanol-onlyextraction followed by LC/MS/MS CoQ10 detection. For example, the totalamount of CoQ10 determined spectroscopically, is at least 5-fold, atleast 10-fold, at least 15-fold, or at least 25-fold greater than usingmethanol-only extraction followed by LC/MS/MS CoQ10 detection.

In certain embodiments of the invention, the second extraction buffercomprises a non-polar solvent. In certain embodiments of the invention,the second extraction buffer comprises an organic solvent. In certainembodiments of the invention, the second extraction buffer comprises asolvent selected from the group consisting of benzene, toluene, xylene,hexane, heptane, octane, cyclohexane, 1, 4-dioxane, chloroform, diethylether, diisopropyl ether, and diisobutyl ether, carbon tetrachloride,dimethyl formamide (DMF), chloromethane, and dichloromethane; or anycombination thereof. In certain embodiments of the invention, the secondextraction buffer comprise an alkane. In certain embodiments of theinvention, the second extraction buffer comprises hexane. In certainembodiments of the invention, the second extraction buffer comprisesacetonitrile. In certain embodiments of the invention, the secondextraction buffer comprises isopropanol.

In certain embodiments of the invention, the first extraction buffercomprises a polar protic solvent. In certain embodiments of theinvention, the first extraction buffer comprises an organic solvent. Incertain embodiments of the invention, the first extraction buffercomprises a solvent selected from the group consisting of formic acid,n-butanol, isopropanol, n-propanol, ethanol, methanol, acetic acid,trichloroacetic acid (TCA), and water; or any combination thereof. Incertain embodiments of the invention, the solvent comprises an alcohol.In certain embodiments of the invention, the alcohol is methanol.

In certain embodiments of the invention, the first extraction buffercomprises a surfactant.

In certain embodiments of the invention, the first extraction buffercomprises a detergent. In certain embodiments of the invention, thedetergent is a mild detergent. In certain embodiments of the invention,the first extraction buffer is an aqueous solution. In certainembodiments of the invention, the first extraction buffer comprises asteroid acid. In certain embodiments of the invention, the steroid acidis a bile acid. In certain embodiments of the invention, the bile acidcomprises an acid selected from the group consisting of taurochloricacid, glycocholic acid, cholic acid, chenodeoxycholic acid, deoxycholicacid, and lithocholic acid; or any combination thereof. In certainembodiments of the invention, the bile acid comprises deoxycholic acid.In certain embodiments of the invention, the deoxycholic acid is a salt.In certain embodiments of the invention, the deoxycholic acid saltcomprises an inorganic ion that is a group I metal. In certainembodiments of the invention, the inorganic ion of the deoxycholic acidsalt comprises an inorganic ion that is a salt selected from the groupconsisting of Li⁺, Na⁺, and K⁺. In certain embodiments of the invention,the inorganic ion of the deoxycholic acid salt comprises an inorganicion that is Na⁺.

In certain embodiments of the invention, the sample comprises abiological sample. In certain embodiments of the invention, thebiological sample is a cell based sample. In certain embodiments of theinvention, the sample comprises a mammalian sample or amphibian sample.In certain embodiments of the invention, the sample comprises a sampleselected from the group consisting of a human sample, a non-humanprimate sample, and a rodent sample. In certain embodiments of theinvention, the sample is a sample from a subject selected from mouse,rat, guinea pig, rabbit, and human.

In certain embodiments of the invention, the spectroscopic analysis iscarried out by using a spectroscopic technique selected from absorptionspectroscopy, fluorescence X-ray spectroscopy, flame spectroscopy,visible spectroscopy, ultraviolet spectroscopy, infrared spectroscopy,near infrared spectroscopy, Raman spectroscopy, coherent anti-StokesRaman Spectroscopy (CARS), nuclear magnetic resonance spectroscopy,photoemission spectroscopy, and Mossbauer spectroscopy. In certainembodiments of the invention, the spectroscopic technique is ultravioletspectroscopy. In certain embodiments of the invention, the ultravioletspectroscopy is performed at one or more wavelengths of 270 nm to 280nm. In certain embodiments of the invention, the ultravioletspectroscopy is performed at one or more wavelengths of 273 nm to 277nm. In certain embodiments of the invention, the ultravioletspectroscopy is performed at a wavelength of 275 nm. In certainembodiments of the invention, the ultraviolet spectroscopy is performedat a wavelength between 270 nm and 280 nm, e.g., 270 nm, 271 nm, 272 nm,273 nm, 274 nm, 275 nm, 276 nm, 277 nm, 278 nm, 279 nm or 280 nm.

In certain embodiments of the invention, the ratio of the volume of thefirst extraction buffer to the volume of the second extraction buffer is5:1 to 1:1 (v/v) (e.g., 5:1, 4:1, 3:1, 2:1, or 1:1; or any rangesbracketed by only those values, e.g., 4:1 to 1:1 or 4:1 to 2:1).

In certain embodiments of the invention, the ratio of the surfactant ordetergent to the volume of the first extraction buffer is 50:1 to 1:1(v/v) (e.g., about 45:1, 40:1, 35:1, 30:1, 25:1, 20:1. 15:1, 10:1, 5:1,2:1, 1:1; or any ranges bracketed by only those values, e.g., 40:1 to10:1, 40:1 to 5:1, 40:1 to 2:1, 40:1 to 1:1, 35:1 to 10:1, 35:1 to 5:1,35:1 to 5:1, 35:1 to 1:1, 25:1 to 10:1, 25:1 to 5:1, 20:1 to 5:1, 15:1to 5:1, 10:1 to 5:1, 10:1 to 2:1, or 10:1 to 1:1).

In certain embodiments of the invention, the sample is heated to 50-100°C., e.g., about 50-100° C., about 55-90° C., about 60-85° C., about60-80° C., about 55-75° C., about 60-70° C., about 62-68° C., about63-67° C., about 64-66° C., or about 65° C., after addition of the firstextraction buffer, second extraction buffer, or both the first andsecond extraction buffer. In certain embodiment, the sample is heated toabout 55° C., about 56° C., about 57° C., about 58° C., about 59° C.,about 60° C., about 61° C., about 62° C., about 63° C., about 64° C.,about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., orabout 70° C. In certain embodiments, the sample is heated to the sametemperature after addition of the first extraction buffer and the secondextraction buffer. In certain embodiments, the sample is heated todifferent temperatures after addition of the first extraction buffer andthe second extraction buffer. In certain embodiments, the sample isheated for a time of at least 5 seconds, at least 10 seconds, at least15 seconds, at least 20 seconds, at least 30 seconds, at least 40seconds, at least 50 seconds, at least 60 seconds, at least 75 seconds,at least 90 seconds, at least 105 seconds, or at least 120 seconds, thatis less than one, but more than zero, seconds to the upper limit of therange.

In certain embodiments of the invention, the sample is heated for thesame amount of time after addition of the first extraction buffer andthe second extraction buffer. In certain embodiments, the sample isheated for different amounts of time after addition of the firstextraction buffer and the second extraction buffer.

In certain embodiments, heating the sample increases the of extractionof CoQ10 by at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, or at least 50% as compared to a control samplethat was not heated.

In certain embodiments of the invention, the sample is mixed bymechanical stirring. In certain embodiments of the invention, the sampleis mixed by sonication. In certain embodiments of the invention, thesample is mixed by magnetic stirring. In certain embodiments, the sampleis mixed after the addition of the first extraction buffer. In certainembodiments, the sample is mixed after the addition of the secondextraction buffer. In certain embodiments, the sample is mixed after theaddition of the first extraction buffer and the second extractionbuffer.

In certain embodiments of the invention, an inorganic salt is added tothe sample after addition of the second extraction buffer. In certainembodiments, the inorganic salt comprises NaCl. In certain embodiments,a saturated brined solution is added to the sample after addition of thesecond extraction buffer. In certain embodiments, the salt is added to afinal concentration of about 1 mM to about 50 mM, that is to aconcentration of about 1 mM to about 10 mM, about 1 mM to about 20 mM,about 1 mM to about 30 mM, about 1 mM to about 40 mM, about 5 mM toabout 50 mM, about 10 mM to about 40 mM, about 20 mM to about 50 mM,about 10 mM to about 25 mM, about 15 mM to about 35 mM; or any range ofvalues bracketed by only those values, e.g., between about 10 mM to 20mM.

In certain embodiments of the invention, the sample is filtered prior tospectroscopic analysis of the second extraction buffer.

In certain embodiments of the invention, the entire method is completedwithin 10 minutes, within 5 minutes, within 1 minute, or within 30seconds.

The invention provides kits for practicing the method of any of thepreceding claims. In certain embodiments, the kit includes at least twoof the following: a first extraction buffer, a second extraction buffer.In another aspect, the kit also comprises CoQ10. In a further aspect,the kit comprises a first extraction buffer, a second extraction bufferand instructions for use.

In another aspect, the invention provides, the invention provides novelmethods of using oxidized and reduced deuterated internal standard forCoQ10. In certain embodiments, the invention provides an LC-MS/MS methodfor determining the amount of CoQ10 and CoQ10H₂.

In some embodiments, the invention provides a method for determining theamount of coenzyme Q10 (CoQ10) in a sample, the method comprising:

a) adding a known amount of deuterated coenzyme Q10 (CoQ10-d6) to thesample;

b) detecting CoQ10 and CoQ10-d6 by mass spectrometry; and

c) determining the amount of detected CoQ10 by comparing it to the knownamount of detected CoQ10-d6.

In some embodiments, the invention also provides a method fordetermining the amount of reduced form of CoQ₁₀ (CoQ₁₀H₂) in a sample,the method comprising:

a) adding a known amount of reduced deuterated coenzyme Q10 (CoQ10H₂-d6)to the sample;

b) detecting CoQ10H₂ and CoQ10H₂-d6 by mass spectrometry; and

c) determining the amount of detected CoQ10H₂ by comparing it to theknown amount of detected CoQ10H₂-d6.

In other embodiments, the invention also provides a method forsimultaneously determining the amounts of CoQ10 and CoQ10H₂ in a sample,the method comprising:

a) adding a known amount of CoQ10-d6 and CoQ10H₂-d6 to the sample;

b) detecting CoQ10, CoQ10H₂, CoQ10-d6 and CoQ10H₂-d6 by massspectrometry; and

c) determining the amount of detected CoQ10 by comparing it to the knownamount of detected CoQ10H₂-d6; and

d) determining the amount of detected CoQ10H₂ by comparing it to theknown amount of detected CoQ10H₂-d6.

In still other embodiments, the invention provides a method fordetermining an extent of CoQ10H₂ oxidation in a sample, the methodcomprising:

a) adding known amounts of CoQ10H₂-d6 and/or CoQ10-d6 to the sample;

b) measuring relative amounts of CoQ10H₂-d6 and CoQ10-d6 in the sampleby mass spectrometry; and

c) comparing theoretical relative amounts of CoQ10H₂-d6 and CoQ10-d6with the relative amounts of CoQ10H₂-d6 and CoQ10-d6 measured in step b.

In other aspects, the invention provides a method for determining anextent of CoQ10H₂ oxidation in a sample, the method comprising:

a) adding known amounts of CoQ10H₂-d6 and/or CoQ10-d6 to the sample;

b) measuring relative amounts of CoQ10H₂-d6 and CoQ10-d6 in the sampleby mass spectrometry at a first time and a second time

c) comparing theoretical relative amounts of CoQ10H₂-d6 and CoQ10-d6present in the same at the first time with the relative amounts ofCoQ10H₂-d6 and CoQ10-d6 present in the same the second time;

wherein the change the relative amount of CoQ10H₂-d6 and CoQ10-d6 at thefirst time and the second time is indicative of the extent of oxidationsustained by the sample.

In some embodiments, CoQ10H₂-d6 is obtained by reacting CoQ10-d6 withone or more reducing agents. In a specific embodiment, the reducingagent is sodium borohydride.

In some embodiments, CoQ10-d6 and/or CoQ10H₂-d6 are added to the sampleimmediately after sample collection.

In one embodiment, the invention provides a method for determining theamount of CoQ10 in a sample, the method comprising:

a) providing the sample;

b) adding a known amount of CoQ10-d6 to the sample;

c) extracting the sample;

d) optionally subjecting the sample to liquid chromatography;

e) detecting CoQ10 and CoQ10-d6 by mass spectrometry; and

f) determining the amount of detected CoQ10 by comparing it to the knownamount of detected CoQ10-d6.

In another embodiment, the invention provides a method for determiningthe amount of CoQ10H₂ in a sample, the method comprising:

a) providing the sample;

b) adding a known amount of CoQ10H₂-d6 to the sample;

c) extracting the sample;

d) optionally subjecting the sample to liquid chromatography;

e) detecting CoQ10H₂ and CoQ10H₂-d6 by mass spectrometry; and

f) determining the amount of detected CoQ10H₂ by comparing it to theknown amount of detected CoQ10H₂-d6.

In yet another embodiment, the invention provides a method fordetermining the amounts of CoQ10 and CoQ10H₂ in a sample, the methodcomprising:

a) providing the sample;

b) adding known amounts of CoQ10-d6 and CoQ10H₂-d6 to the sample;

c) extracting the sample;

d) optionally subjecting the sample to liquid chromatography;

e) detecting CoQ10, CoQ10-d6, CoQ10H₂ and CoQ10H₂-d6 by massspectrometry;

f) determining the amount of detected CoQ10 by comparing it to the knownamount of detected CoQ10-d6; and

g) determining the amount of detected CoQ10H₂ by comparing it to theknown amount of detected CoQ10H₂-d6.

In some embodiments, step c comprises adding an extraction buffer to thesample, e.g., a first extraction buffer. In a specific embodiment, theextraction buffer, e.g., the first extraction buffer, comprises1-propanol. In other embodiments, the extraction buffer comprisesisopropanol, methanol, ethanol, acetonitrile or acetone. In oneembodiment, the extraction with the extraction buffer, e.g., 1-propanol,is followed by a mass spectroscopic analysis of the solvent used forextracton.

In a further embodiment, step c further comprises adding a secondextraction buffer that results in phase separation of the sample. In oneembodiment, step c comprises:

i. adding a first extraction buffer and a second extraction buffer tothe sample;

ii. mixing the sample; and

iii. using the second extraction layer for subsequent steps.

In one embodiment, step c comprises:

i. adding a first extraction buffer to the sample;

ii. heating and mixing the sample;

iii. adding a second extraction buffer to the sample that results inphase separation of the sample;

iv. heating and mixing the sample;

v. cooling the sample to ambient temperature; and

vi. using the second extraction buffer layer for subsequent steps.

In some embodiments, steps b-d are carried out in reduced light. In someembodiments, steps b-d are carried out using pre-cooled solvents. Insome embodiments, steps b-d are carried using pre-cooled cryo-block. Insome embodiments, the cryo-block is pre-cooled for about 1 hour to about48 hours, e.g., about 1 hour, about 2 hours, about 3 hours, about 4hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about9 hours, about 10 hours, about 11 hours, about 12 hours, about 14 hours,about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 28hours, about 32 hours, about 36 hours, about 40 hours, about 44 hours orabout 48 hours.

In certain embodiments, the sample is a biological sample. In a specificembodiment, the biological sample is plasma. In another specificembodiment, the biological sample is serum. In another specificembodiment, the biological sample is a tissue. In yet anotherembodiment, the biological sample is a bodily fluid, e.g., blood, urine,saliva or nasal mucus. In one embodiment, the biological sample is ahuman sample.

In some embodiment, the invention also provides the kits for carryingout the mass spectrometric methods of the invention. In some embodiment,the kit comprises an extraction buffer, e.g., 1-propanol. In someembodiments, the kit comprises CoQ10-d6. In another embodiment, the kitcomprises CoQ10-d6 and one or more reducing agents. In a specificembodiment, the reducing agent is sodium borohydride. In a furtherembodiment, the kit comprises an extraction buffer, e.g., 1-propanol,CoQ10-d6 and a reducing agent, e.g., sodium borohydride.

Other embodiments are provided infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a representative standard curve of OD₂₇₅ values graphedagainst the indicated concentrations of CoQ10 in each of acetonitrile,2-propanol, and hexane. The equations for each line is provided.

FIG. 2 shows a representative standard curve of OD₂₇₅ values graphedagainst the indicated concentrations of CoQ10 in hexane.

FIG. 3A-E show graphs of representative results of CoQ10 concentrations(pM/cell) in three replicates, and averages, of (A) untreated HepG2cells; (B) HepG2 cells treated with CoQ10 at 50 μM in propanol; (C)HepG2 cells treated with CoQ10 at 100 μM in propanol; (D) HepG2 cellstreated with CoQ10 at 50 μM in a CoQ10 delivery formulation; or (E)HepG2 cells treated with CoQ10 at 100 μM in a CoQ10 deliveryformulation; cultured in the presence of CoQ10 for the times indicatedand extracted using the methods of the invention.

FIG. 4 shows standard curves for CoQ10- and CoQ10H₂, with samplesprepared using hexane as a diluent and hexane-reconstituted CoQ10- andCoQ10H₂ stocks.

FIG. 5 shows standard curves for CoQ10- and CoQ10H₂, with samplesprepared using water as a diluent and methanol-reconstituted CoQ10- andCoQ10H₂ stocks.

FIG. 6 shows a standard curve and representative chromatograms for totalCoQ10 in plasma samples.

FIG. 7 shows representative chromatograms for total CoQ10 in cellsupernatants.

FIG. 8 shows a standard curve and representative chromatograms for totalCoQ10 in nasal wash.

FIG. 9 shows a standard curve and a representative chromatogram fortotal CoQ10 in tissue samples.

FIG. 10 shows a standard curves for CoQ10- and CoQ10H₂ in serum samples.

FIG. 11 shows representative chromatograms for CoQ10- and CoQ10H₂ inserum samples.

FIG. 12 shows a standard curves for CoQ10- and CoQ10H₂ in plasmasamples.

FIG. 13 shows representative chromatograms for CoQ10- and CoQ10H₂ inplasma samples.

DETAILED DESCRIPTION

The present invention relates to methods for rapidly and efficientlyextracting CoQ10 from a biological sample, preferably a cell basedsample, to detect CoQ10, preferably quantitatively, in the sample. Themethods can be readily adapted for use in a high throughputand/automated screening method.

For purposes of optimizing readability and to facilitate understandingof the invention as described herein, it may be beneficial to considerthe following definition of terms and phrases as used herein.

I. Definitions

The detection methods provided herein can be used for the detection ofCoenzyme Q10 (CoQ10) and its structural variants. For example, CoQ10 canexist in a fully oxidized form (ubiquinone), a partially oxidized form(ubisemiquinone), and fully reduced form (ubiquinol). Further, althoughCoQ10 as shown above has 10 isoprenoid units, the methods providedherein can be used for the detection of compounds structurally similarto CoQ10 with about 8-12 (e.g., 8, 9, 10, 11, 12) isoprenoid units, asthe ring structure is detected using the spectrophotometric methodspreferred herein.

“Total CoQ10”, as used herein, refers to the total amount of oxidizedand reduced CoQ10 present in a sample.

A “solvent” as used herein is understood typically as a liquid, thatdissolves a solid or liquid, resulting in a solution. The solute issoluble in a certain volume of solvent at a specified temperature. Asused herein, the solvent does not need to completely solvate all of thesample as long as the non-dissolved components do not interfere with theextraction and detection of CoQ10 in the sample. For example, when wholecell based samples are used, precipitates including proteins and/ornucleic acids can be formed.

Solvents can be subdivided into various groups based on chemicalcharacteristics, solvation/reaction mechanisms, overall charge, chargedistribution, etc. For example, solvents can be grouped into non-polarsolvents and polar solvents, which can be further subdivided into polaraprotic solvents and polar protic solvents.

The solvents set forth below are ordered by increasing polarity asdefined by the dielectric constant. The properties of solvents that aregreater than those of water are bolded.

TABLE 1 Organic Solvents Boiling Dielectric Dipole Solvent ChemicalFormula point constant Density moment (D) Non-Polar Solvents HexaneCH₃—CH₂—CH₂—CH₂—CH₂—CH₃ 69° C. 2.0 0.655 g/ml 0.00 D Benzene C₆H₆ 80° C.2.3 0.879 g/ml 0.00 D Toluene C₆H₅—CH₃ 111° C.  2.4 0.867 g/ml 0.36 D1,4-Dioxane /—CH₂—CH₂—O—CH₂—CH₂—O—\ 101° C.  2.3 1.033 g/ml 0.45 DChloroform CHCl₃ 61° C. 4.8 1.498 g/ml 1.04 D Diethyl etherCH₃CH₂—O—CH₂—CH₃ 35° C. 4.3 0.713 g/ml 1.15 D Polar Aprotic SolventsDichloromethane CH₂Cl₂ 40° C. 9.1 1.3266 g/ml  1.60 D (DCM)Tetrahydrofuran (THF) /—CH₂—CH₂—O—CH₂—CH₂—\ 66° C. 7.5 0.886 g/ml 1.75 DEthyl acetate CH₃—C(═O)—O—CH₂—CH₃ 77° C. 6.0 0.894 g/ml 1.78 D AcetoneCH₃—C(═O)—CH₃ 56° C. 21 0.786 g/ml 2.88 D DimethylformamideH—C(═O)N(CH₃)₂ 153° C.  38 0.944 g/ml 3.82 D (DMF) Acetonitrile (MeCN)CH₃—C≡N 82° C. 37 0.786 g/ml 3.92 D Dimethyl sulfoxide CH₃—S(═O)—CH₃189° C.  47 1.092 g/ml 3.96 D (DMSO) Polar Protic Solvents Formic acidH—C(═O)OH 101° C.  58  1.21 g/ml 1.41 D n-Butanol CH₃—CH₂—CH₂—CH₂—OH118° C.  18 0.810 g/ml 1.63 D Isopropanol (IPA) CH₃—CH(—OH)—CH₃ 82° C.18 0.785 g/ml 1.66 D n-Propanol CH₃—CH₂—CH₂—OH 97° C. 20 0.803 g/ml 1.68D Ethanol CH₃—CH₂—OH 79° C. 30 0.789 g/ml 1.69 D Methanol CH₃—OH 65° C.33 0.791 g/ml 1.70 D Acetic acid CH₃—C(═O)OH 118° C.  6.2 1.049 g/ml1.74 D Water H—O—H 100° C.  80 1.000 g/ml 1.85 D

“Non-polar solvents” are solvents in which there is (almost) no polarityin the bonds, or, in which the bonds are symmetrically arranged,resulting in balanced pull of charge in all directions. For example,chloroform has relatively strong polar bonds, however, the trigonalplanar arrangement of three polar bonds with equal dipole moments makesthe molecule non-polar. Alternatively, in alkanes, the bonds have weakdipole moments and almost no polarity in the bonds, making themnon-polar. Non-polar solvents include, but are not limited to, alkanes,benzene, toluene, xylenes, hexanes, heptanes, octanes, cyclohexane, 1,4-dioxane, chloroform, diethyl ether, diisopropyl ether, and diisobutylether.

Alkanes (also known as paraffins or saturated hydrocarbons) are chemicalcompounds that consist only of the elements carbon (C) and hydrogen (H)(i.e., hydrocarbons), wherein these atoms are linked togetherexclusively by single bonds (i.e., they are saturated compounds).Alkanes belong to a homologous series of organic compounds in which themembers differ by a constant relative molecular mass of 14. Each carbonatom must have 4 bonds (either C—H or C—C bonds), and each hydrogen atommust be joined to a carbon atom (H—C bonds). As a result, alkanes aretypically very stable, and have little biological activity. A series oflinked carbon atoms is known as the carbon skeleton or carbon backbone.In general, the number of carbon atoms is often used to define the sizeof the alkane (e.g., C₂-alkane).

The simplest possible alkane is methane, CH₄. There is theoretically nolimit to the number of carbon atoms that can be linked together, theonly limitation being that the molecule is saturated, and is ahydrocarbon. Saturated oils and waxes are examples of larger alkaneswhere the number of carbons in the carbon backbone tends to be greaterthan 10. As used herein, lower alkanes have 1 to 6 carbon atoms. Higheralkanes have at least 7 carbon atoms, preferably 7 to 12 carbon atoms.As used herein, middle alkanes have 4 to 8 carbons, or in certainembodiments, 5, 6, or 7 carbon atoms.

Straight-chain alkanes are sometimes indicated by the prefix n- (fornormal) where a non-linear isomer exists. Although this is not strictlynecessary, the usage is still common in cases where there is animportant difference in properties between the straight-chain andbranched-chain isomers, e.g., n-hexane or 2- or 3-methylpentane. Themembers of the series (in terms of number of carbon atoms) are named asfollows: methane, CH₄— one carbon and four hydrogen; ethane, C₂H₆— twocarbon and six hydrogen; propane, C₃H₈— three carbon and 8 hydrogen;butane, C₄H₁₀— four carbon and 10 hydrogen; pentane, C₅H₁₂— five carbonand 12 hydrogen; and hexane, C₆H₁₄— six carbon and 14 hydrogen. As usedherein, unless the n-prefix is used, alkanes are understood to refer toone or more carbon compounds having the indicated number of carbons, andcan include mixed populations of the alkane identified (e.g., hexane isunderstood to include any of n-hexane, 2-methyl pentane,3-methylpentane, and cyclohexane; and any combination thereof with anyratios of the various hexanes).

“Protic solvents” as used herein, solvate anions strongly via hydrogenbonding. Protic solvents have an acidic hydrogen, although they may bevery weak acids. More generally, any molecular solvent which containsdissociable H⁺, is called a polar protic solvent. The molecules of suchsolvents can donate an H⁺ (proton). Protic solvents stabilize ions bystabilizing unshared electron pairs with cations, and by hydrogenbonding with anions.

“Polar protic solvents” favor the SN1 reaction mechanism as the solventshave a hydrogen atom bound to an oxygen, as in a hydroxyl group, or anitrogen, as in an amine group. Polar protic solvents, as used herein,tend to have high dielectric constants and high polarity. Polar proticsolvents include alcohols.

An “alcohol” is any organic compound in which a hydroxyl functionalgroup (—OH) is bound to a carbon atom, in which the carbon atom isusually connected to other carbon or hydrogen atoms. The hydroxyl (OH)functional group in an alcohol molecule, with the dissociable proton inthe —OH group, makes the alcohol a polar protic solvent.

Alcohols include, for example, acyclic alcohols have the general formulaof C_(n)H_(2n+1)OH where n≥1. The suffix -ol appears in the IUPACchemical name of all substances where the hydroxyl group is thefunctional group with the highest priority; in substances where a higherpriority group is present the prefix hydroxy—will appear in the IUPACname. The suffix -ol in non-systematic names (such as paracetamol orcholesterol) also typically indicates that the substance includes ahydroxyl functional group and so can be termed an alcohol, but manysusbtances (such as citric acid, lactic acid, or sucrose) contain one ormore hydroxyl functional groups. As used herein, alcohols can becharacterized by the number of carbons present, with lower alcoholshaving 1-6 carbons (i.e., n=1-6 in the above formula for acyclicalcohols), middle alcohols having 4-8 carbons, and upper alcohols havingat least 7 carbons, preferably having 7-12 carbons. Some alcoholsinclude, but are not limited to, methanol (CH₃OH), ethanol (C₂H₅OH),propanol (C₃H₇OH), and pentanol (C₅H₁₁OH). As used herein, unless then-prefix is used, alcohols are understood to refer to one or more carboncompounds having the indicated number of carbons, and can include mixedpopulations of the alcohol identified.

“Polar aprotic solvents” are solvents that share ion dissolving powerwith protic solvents but lack an acidic hydrogen. Therefore polaraprotic solvents do not solvate anions, which can inhibit subsequentchemical reactions. These solvents generally have intermediatedielectric constants and polarity. Typically aprotic solvents do notdisplay hydrogen bonding and do not have an acidic hydrogen, but areable to stabilize ions. Nucleophiles are more reactive in aprotic thanprotic solvents.

“Organic solvents” are solvents that include at least one carbon atom.

“Detergents” as used herein are small amphipathic molecules that tend toform micells in water. Detergents are typically classified according totheir hydrophilic/hydrophobic character and ionic groups. Detergents areuseful for permeablization/solubilization of membranes,decellularization of organs or tissues, maintaining stability ofpurified proteins by limiting aggregation, and dissolving lipids andother hydrophobic molecules. Examples of detergents can be found, forexample, in 2010 McCutcheon's Emulsifiers & Detergents North Americanedition (McCutcheon's Emulsifiers and Detergents), incorporated hereinby reference. A “mild detergent” is typically considered a detergentthat does not disrupt the structure of a protein in solution. Thisallows the determination of the function of the protein. A “milddetergent” typically has a ratio of polar/nonpolar side chain whichfavors one or the other (e.g. large head group, short aliphatic chain).In general, non-ionic and zwitterionic detergents are more mild thanionic detergents. Of the ionic detergents, bile acids and bile salts areconsidered to be mild detergents. Anionic detergents are not milddetergents.

Ionic detergents are characterized by their charged hydrophilicheadgroups. Ionic detergents can be anionic or cationic. Ionicdetergents tend to disrupt both inter- and intra-molecularprotein-protein interactions. Cationic detergents include, but are notlimited to, cationic surfactant solution comprising a selectedquaternary amine. The selected quaternary amines are produced throughthe reaction of a quaternary amine hydroxide and an acid of the groupconsisting of phosphoric, sulfuric, formic, acetic, propionic, oxalic,malonic, succinic and citric, e.g., an alkyltrimethylammonium or analkylbenzyldimethylammonium, where the alkyl group contains 12, 14, 16or 18 carbons. Ionic detergents include deoxycholic acid, sodium dodecylsulfate (SDS), and hexadecyltrimethylammonium bromide (CTAB).

Non-ionic (or zwitterionic) detergents are characterized by their (net)uncharged, hydrophilic headgroups. They are based on polyoxyethyleneglycol (i.e. TWEEN®, TRITON®, and BRIJ® series), CHAPS®, glycosides(i.e. octyl-thio-glucoside, maltosides), bile acids such as deoxycholicacid (DOC), Lipids (HEGAs®), or phosphine oxides (e.g., inorganicphosphorus compounds such as phosphoryl trichloride (Cl₃P═O) ororganophosphorus compounds with the formula OPR₃, where R=alkyl oraryl). Other non-ionic detergents include ethoxylated fatty alcoholethers and lauryl ethers, ethoxylated alkyl phenols, octylphenoxypolyethoxy ethanol compounds, modified oxyethylated and/or oxypropylatedstraight-chain alcohols, polyethylene glycol monooleate compounds,polysorbate compounds, and phenolic fatty alcohol ethers.

“Surfactants” as used herein, are understood as compounds that lower thesurface tension of a liquid, allowing easier spreading, and lowering ofthe interfacial tension between two liquids, or between a liquid and asolid. Surfactants additionally may act as one or more of detergents,wetting agents, emulsifiers, foaming agents, and dispersants.

The term surfactant is a blend of surface active agent. Surfactants areusually organic compounds that are amphiphilic, meaning they containboth hydrophobic groups (their tails) and hydrophilic groups (theirheads). Therefore, a surfactant molecule contains both a water insoluble(or oil soluble component) and a water soluble component. Surfactantmolecules migrate to the water surface, where the insoluble hydrophobicgroup may extend out of the bulk water phase, either into the air or, ifwater is mixed with an oil, into the oil phase, while the water solublehead group remains in the water phase. This alignment and aggregation ofsurfactant molecules at the surface acts to alter the surface propertiesof water at the water/air or water/oil interface.

Bile acids are steroid acids that solvate fats by the formation ofmicells. Bile acids are useful as both detergents and surfactants. Bileacid refers to the protonated (—COOH) form. Bile salt refers to thedeprotonated or ionized (—COO⁻) form and are typically conjugated toglycine or taurine. Conjugated bile acids are more efficient atemulsifying fats at neutral pH, because they are more ionized thanunconjugated bile acids. Bile salts are frequently used as detergents tosolvate lipids in protein purification and histochemical methods. Bilesalts can also be used as surfactants. Unless otherwise clear fromcontext, as use herein bile acids are understood to include bile acidsalts. Bile acids include, but are not limited to, cholic acid,chenodeoxycholic acid, glycholic acid, taurocholic acid, deoxycholicacid, and lithocholic acid. In research deoxycholic acid is used as adetergent for the isolation of membrane associated proteins. Sodiumdeoxycholate, the sodium salt of deoxycholic acid, is often used as abiological detergent to lyse cells and solubilise cellular and membranecomponents.

“Critical micellar concentration” or “CMC” refers to both an intrinsicproperty of amphipathic molecules capable of forming micells, such assurfactants or detergents, and of the amphipathic molecules in solutionin the amount of bile acid necessary to function in the spontaneous anddynamic formation of micelles. Upon introduction of surfactants (or anyamphipathic molecules capable of forming micells) into the system theyinitially partition into the interface, reducing the system free energyby a) lowering the energy of the interface (calculated as area×surfacetension) and b) by removing the hydrophobic parts of the surfactant fromcontact with water. Subsequently, when the surface coverage by thesurfactants increases and the surface free energy (surface tension)decreases, the surfactants aggregate into micelles, thereby decreasingthe system's free energy by decreasing the contact area of hydrophobicparts of the surfactant with water. Upon reaching CMC, any furtheraddition of surfactants will increase the number of micelles. In themethods provided herein, surfactants are typically used at aconcentration such that the critical micell concentration is reached inthe sample.

As used herein, “salts” are ionic compounds in which the proportions ofthe ions are such that the electric charges cancel out, so that the bulkcompound is electrically neutral. “Inorganic salt” includes saltsinclude, for example, oxides, carbonates, sulfates, and halides. Thehalides include fluoride (F⁻), chloride (Cl⁻), bromide (Br⁻), iodide(I⁻) and astatide (At⁻). Inorganic halide salts include, for example,sodium chloride (NaCl), potassium chloride (KCl), potassium iodide (KI),lithium chloride (LiCl), copper(II) chloride (CuCl₂), silver chloride(AgCl), and chlorine fluoride (ClF). As used herein, a “brined solution”is an inorganic salt solution.

As used herein, a “saturated solution” is understood by its typicaldefinition in physical chemistry as a solution of a substance candissolve no more of that substance and additional amounts of it willappear as a precipitate. This point of maximum concentration, thesaturation point, depends on the temperature of the liquid as well asthe chemical nature of the substances involved. As used herein,saturation will be determined at ambient temperature.

As used herein, a “first extraction buffer” includes a polar proticsolvent and/or an organic solvent. Polar protic solvents for use in afirst extraction buffer include, but are not limited to, alcoholsincluding acyclic alcohols, such as butanol, propanol (i.e, isopropanoland/or n-propanol), ethanol, methanol, hexanol, octanol, etc.; formicacid, acetic acid, and water. As used herein, the first solvent promotesat least one of: disrupting structure of proteins, disrupting cellstructure e.g., by creating holes in the cell membrane, promoting atleast a limited extraction of proteins, and promoting proteinprecipitation. As used herein, the first solvent promotes at least twoof: disrupting structure of proteins, disrupting cell structure e.g., bycreating holes in the cell membrane, promoting at least a limitedextraction of proteins, and promoting protein precipitation. As usedherein, the first solvent promotes at least three of: disruptingstructure of proteins, disrupting cell structure e.g., by creating holesin the cell membrane, promoting at least a limited extraction ofproteins, and promoting protein precipitation. As used herein, the firstsolvent promotes all of: disrupting structure of proteins, disruptingcell structure e.g., by creating holes in the cell membrane, promotingat least a limited extraction of proteins, and promoting proteinprecipitation. The first extraction buffer is different from the secondextraction buffer. In certain embodiments, the first extraction bufferis not methanol.

As used herein, a “second extraction buffer” includes a non polarsolvent, for example an organic solvent. The second extraction buffercan include, but is not limited to, benzene, toluene, xylenes, hexane,heptane, octane, hexanes, cyclohexane, 1, 4-dioxane, chloroform, diethylether, diisopropyl ether, and diisobutyl ether. The second extractionbuffer can also include an alkane such as hexane. In certainembodiments, the second extraction buffer is a polar aprotic solvent,such as acetonitrile. As used herein, the second solvent promotespartitioning of CoQ10 away from the first extraction buffer. The secondextraction buffer is different from the first extraction buffer.

The term “sample,” as used herein, is used in its broadest sense. A“biological sample,” as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly living thingthat can be solubilized in a first extraction buffer optionallycontaining a surfactant or detergent. Such living things include, butare not limited to, mammals, humans, non-human primates, mice, rats,monkeys, dogs, rabbits, and other animals; plants; single celledorganisms such as yeast and bacteria. Such substances include, but arenot limited to, blood, (e.g., whole blood), plasma, serum, urine,amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes, andspleen, e.g., from resected tissue or biopsy samples; and cellscollected, e.g. by centrifugation, from any bodily fluids; and primaryand immortalized cells and cell lines. Samples can include fresh samplesand historical samples. As used herein, a “cell based sample” isunderstood as a sample wherein substantially all (e.g., at least 90%, atleast 95%, at least 98%, at least 99%) of the CoQ10 present in thesample for detection is present inside cells of the sample (i.e., not inserum, extracellular fluid, cell culture media). In certain embodiments,the methods provided herein are for the detection of CoQ10 in cell basedsamples.

“Spectrophotometry” or “spectroscopic analysis” as used herein andunderstood in chemistry is the quantitative measurement of thereflection or transmission properties of a material as a function ofwavelength. As used herein, spectrophotometry is related to transmissionand detection of visible light, near-ultraviolet, and near-infrared.Preferably, the spectrophotometric methods are used to determine theconcentration of a component of a mixture that absorbs light at aparticular wavelength, without separating the component to be detectedfrom the other components of the mixture, e.g., by size separation orprecipitation. Preferably, the spectrophotometric methods providedherein do not include or require isolation of the CoQ10 from the celllysate using size based exclusion or separation methods (e.g.,chromatography or mass spectrometry) to allow detection of CoQ10 in thesample.

Spectroscopic analysis is selected from absorption spectroscopy,fluorescence X-ray spectroscopy, flame spectroscopy, visiblespectroscopy, ultraviolet spectroscopy, infrared spectroscopy, nearinfrared spectroscopy, Raman spectroscopy, coherent anti-Stokes RamanSpectroscopy (CARS), nuclear magnetic resonance spectroscopy,photoemission spectroscopy, and Mossbauer spectroscopy. CoQ10 ispreferably detected at a wavelength near or at 275 nm (e.g., 270-280 nm;272-278 nm; 274-276 nm), using ultraviolet spectroscopy. In someembodiments, CoQ10 is detected at any wavelength between 270 nm and 280nm, e.g., 270 nm, 271 nm, 272 nm, 273 nm, 274 nm, 275 nm, 276 nm, 277nm, 278 nm, 279 nm or 280 nm.

As used herein, “isolation” of CoQ10 as used herein is understood tomean that in the solvent, the CoQ10 is at least 80%, 85%, 90%, 95%, 97%,98%, or 99% free of materials that naturally occur with CoQ10 (e.g.,cellular components, biological sample components).

“Determining the amount” as used herein is performing a step to detectthe presence, or absence, of an analyte, e.g., CoQ10, in a sample. Incertain embodiments, the amount of analyte determined to be in a samplecan be none or below the limit of detection of the method. In certainembodiments, the amount of CoQ10 may be greater than the lineardetection range of the method. In such cases, the sample can be dilutedin an appropriate buffer prior to spectrophotometric analysis.

The term “control sample,” as used herein, refers to any clinicallyrelevant comparative sample, including, for example, a sample from ahealthy subject not afflicted with cancer or other disease, a samplefrom a subject having a less severe or slower progressing cancer orother disease than the subject to be assessed, a sample from a subjecthaving some other type of cancer or disease, a sample from a subjectprior to treatment, a sample of non-diseased tissue (e.g., non-tumortissue), a sample from the same origin and close to the tumor site, andthe like. A control sample may include a sample derived from one or moresubjects. A control sample may also be a sample made at an earlier timepoint from the subject to be assessed. For example, the control samplecould be a sample taken from the subject to be assessed before the onsetof the cancer, or other disease, at an earlier stage of disease, orbefore the administration of treatment or of a portion of treatment. Acontrol sample can be a purified sample, a chemical compound (e.g.,CoQ10), protein, and/or nucleic acid provided with a kit. Such controlsamples can be diluted, for example, in a dilution series to allow forquantitative measurement of analytes in test samples. The control samplemay also be a sample from an animal model, or from a tissue or celllines derived from the animal model of disease. As the level of CoQ10varies between tissues, the control can be a tissue specific control.The level of CoQ10 in a control sample that consists of a group ofmeasurements may be determined, e.g., based on any appropriatestatistical measure, such as, for example, measures of central tendencyincluding average, median, or modal values.

The term “control level” refers to an accepted or pre-determined levelof CoQ10, either a cut-off value, or a series of values used to generatea standard curve, which is used to compare with the spectrophotometricreading used to determine the level of CoQ10 a sample, e.g. a samplederived from a subject. For example, in one embodiment, the controllevel of CoQ10 is based on the level of CoQ10 in sample(s) from asubject(s) having or suspected of having a particular disease. Inanother embodiment, the control level of CoQ10 is based on the level ina sample from a subject(s) having a particular rate of diseaseprogression. In another embodiment, the control level of CoQ10 is basedon the level of CoQ10 in a sample(s) from an unaffected, i.e.,non-diseased, subject(s), i.e., a subject who has not been diagnosed oris not expected to have a particular disease. In yet another embodiment,the control level of CoQ10 is based on the level of CoQ10 in a samplefrom a subject(s) prior to the administration of a therapy. In anotherembodiment, the control level of CoQ10 is based on the level of CoQ10 ina sample(s) from a subject(s) having a disease or condition that is notcontacted with a test compound. In one embodiment, the control is astandardized control, such as, for example, a control which ispredetermined using an average of the levels of CoQ10 from a populationof subjects having no particular disease or condition, or diagnosed witha particular disease or condition, or in normal tissue adjacent toabnormal tissue (e.g., normal tissue adjacent to tumor tissue). Controllevels can also be ranges, e.g., levels typically detected in normal orcontrol tissues; or control values can include values at the ends ofnormal ranges, for example, the amount detected relative to the upperlevel of normal or the lower level of normal.

“Baseline” refers to the level of CoQ10 upon patient entrance into astudy or at the initiation of treatment and is used to distinguish fromlevels of CoQ10 the patient might have during or after treatment.Baseline levels can be used as control levels.

As used herein, “changed as compared to a control” sample or subject isunderstood as having a level of CoQ10 to be detected at a level that isstatistically different than a sample from a normal, untreated, orcontrol sample. Control samples include, for example, cells in culture,particularly untreated or vehicle treated cells, one or more laboratorytest animals, or one or more human subjects. Methods to select and testcontrol samples are within the ability of those in the art. An analytecan be a naturally occurring substance that is characteristicallyexpressed or produced by the cell or organism (e.g., CoQ10, an antibody,a protein) or a substance produced by a reporter construct (e.g.,β-galactosidase or luciferase). Depending on the method used fordetection the amount and measurement of the change can vary. Changed ascompared to a control reference sample can also include a change in oneor more signs or symptoms associated with or diagnostic of disease,e.g., cancer. Determination of statistical significance is within theability of those skilled in the art, e.g., the number of standarddeviations from the mean that constitute a positive result.

“Mixing” and the like, as used herein, is understood as combining orblending of components, e.g., two or more liquids. Mixing can becontinuous or intermittent. Mixing can be performed, for example, byagitation of the container in which the components are contained (i.e.,mechanical stirring), by magnetic stirrer, by sonication, by vortexing,or any other method that results in efficient commingling of thecomponents. In preferred embodiments, mixing methods that do not createbubbles that can interfere with later detection methods, particularly inthe presence of a surfactant or detergent, are preferred. In certainembodiments, antifoaming agents can be included to reduce or preventfoaming as long as the antifoaming agent does not interfere withdetection of CoQ10.

As used herein, “ambient temperature” is understood to be ambienttemperature in a laboratory, e.g., typically about 15° C. to about 30°C., preferably about 18° C. to about 25° C. As used herein, cooling to“ambient temperature” is cooling to a temperature that allows for phaseseparation of the aqueous and organic layers of the treated samples ofthe invention. Cooling can be performed using a water bath, temperatureblock, a blower, or other device; or simply by allowing the samples toreach ambient temperature.

As used herein, “heating” the sample is understood as increasing thesample to a temperature of about 50° C. to about 100° C., about 55° C.to about 90° C., 60° C. to about 80° C., about 60° C. to about 70° C.,about 62° C. to about 68° C., about 63° C. to about 67° C., or about 65°C.; or a range bracketed by any of the values provided. Heating can beperformed using a water bath, temperature block, a blower, incubator, orother device.

Extraction efficiency is determined as the amount of CoQ10 extractedusing the methods of the invention relative to the amount of CoQ10extracted using methanol alone, the method routinely used for CoQ10extraction prior to detection using mass spectrophotometric methods forCoQ10. In certain embodiments, after extraction, the CoQ10 is detectedusing spectrophotometry. In certain embodiments, after extraction, theCoQ10 is detected using LC/MS/MS. In certain embodiments, afterextraction, the CoQ10 is detected using spectrophotometry for onesample, and LC/MS/MS for the other sample. Extraction efficiency isexpressed as increased fold efficiency over extraction methods usingmethanol alone, e.g., about a 2-, 3-, 4-, 5-, 7-, 10-, 15-, 20-, 25-,30-, 40-, 50-, 75-, or 100-fold or more increase in efficiency.

As used herein, “automation” is understood as a process carried out by amachine and do not require manual sample or reagent handling. It isunderstood that some sample and device preparation is required to allowfor processing of samples by machine, e.g., determining the appropriateamount of sample for analysis, placing samples in appropriate containersto allow for processing, loading reagents into the machine, programmingthe machine, and data analysis. As used herein, the process is automatedif all of the claimed steps are automated.

As used herein, an isotopically labeled analog of the oxidized form ofcoenzyme Q10 (CoQ10-d6) is the compound having the following structuralformula:

As used herein, an isotopically labeled analog of the reduced form ofcoenzyme Q10 (CoQ10H₂-d6) is the compound having the followingstructural formula:

In some embodiments, CoQ10H₂-d6 is synthesized by reacting CoQ10-d6 withone or more reducing agents. In some embodiments, the reducing agent issodium borohydride (NaBH₄).

As used herein, “internal standard” is understood as a chemicalsubstance that is added in a known amount directly to each samplecontaining an analyte. The amount of analyte present is then determinedrelative to the internal standard as a calibrant.

In some embodiments, the internal standard is an isotopically labeledinternal standard, and is an isotopically labeled version of the analytemolecule. The mass spectrometric signal produced by the analyte differsfrom the mass spectrometric signal produced by the isotopically labeledstandard, the difference being dependent on the type and the number ofthe isotope atoms incorporated into the isotopically labeled version ofthe analyte. In some embodiments, CoQ10-d6 is an isotopically labeledversion of CoQ10 that functions as an internal standard. In someembodiments CoQ10H₂-d6 is an isotopically labeled version of CoQ10H₂that functions as an internal standard.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrometric instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; andMerchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N.sub.2 gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M. Because the photon energy typically is justabove the ionization potential, the molecular ion is less susceptible todissociation. In many cases it may be possible to analyze sampleswithout the need for chromatography, thus saving significant time andexpense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH⁺. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M⁺ and MH⁺ is constant. Drug compounds in protic solvents areusually observed as MH⁺, whereas nonpolar compounds such as naphthaleneor testosterone usually form M. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions. As used herein,the term “desorption” refers to the removal of an analyte from a surfaceand/or the entry of an analyte into a gaseous phase. Laser diode thermaldesorption (LDTD) is a technique wherein a sample containing the analyteis thermally desorbed into the gas phase by a laser pulse. The laserhits the back of a specially made 96-well plate with a metal base. Thelaser pulse heats the base and the heat causes the sample to transferinto the gas phase. The gas phase sample may then be drawn into anionization source, where the gas phase sample is ionized in preparationfor analysis in the mass spectrometer. When using LDTD, ionization ofthe gas phase sample may be accomplished by any suitable technique knownin the art, such as by ionization with a corona discharge (for exampleby APCI).

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring” or “multiple reaction monitoring” is a detectionmode for a mass spectrometric instrument in which a precursor ion andone or more fragment ions are selectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of less than 20% and an accuracy of 80% to 120%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three times the RSD ofthe mean at the zero concentration.

As used herein, “amber vial” is a vial made from amber glass. In someembodiments, the amber vial is used to protect the sample containedtherein from light.

As used herein, “cryo-block” is a test-tube holder that can keep thetest tubes cold for prolonged periods of time.

As used herein, “kits” include two or more reagents to practice themethod of the invention in appropriate packaging. For example, a kit caninclude any one or more of a lysis buffer, a first extraction buffer, asecond extraction buffer, and a detergent or surfactant, either inliquid or lyophilized form. In certain embodiments, the kit can includea surfactant and/or CoQ10 for use as a control, e.g., to allow forgeneration of a standard curve. Kits can also include instructions forperforming the methods of the invention.

As used herein, “obtaining” is understood herein as manufacturing,purchasing, or otherwise coming into possession of.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear; however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition.

Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. “A”,“an”, and “the” should be understood to include both plural and singularunless stated otherwise or obvious from context. As used herein, “or”should be understood as being inclusive unless stated otherwise orobvious from context.

Also, terms such as “element” or “component” encompass both elements andcomponents comprising one unit and elements and components that comprisemore than one subunit unless specifically stated otherwise.

The term “including” and “comprising”, and the like, are not limitingand should be understood to allow the inclusion of other components orsteps. The term “such as” is used herein to mean, and is usedinterchangeably, with the phrase “such as but not limited to”.

As used herein, “consisting essentially of” and the like is understoodto limit the compound, method, or kit to the specified materials orsteps “and those that do not materially affect the basic and novelcharacteristic(s)” of the invention. For example, extraction methods canconsist essentially of extraction with a first extraction buffer, asecond extraction buffer, a detergent, and optionally a salt with nofurther extraction or purification steps. However, other steps such astransferring or mixing samples may be included.

Unless otherwise clear from context, all values herein can be understoodto be modified by the term “about”. The amount of variation toleratedwill depend on the specific value, but is typically considered to bewithin two standard deviations of the mean. “About” can be understood tobe a variation of up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%,or 0.01%. Ranges provided herein are understood to include all of thevalues within the range, or any subset of ranges or values within therange. For example, 1-10 is understood to include 1, 2, 3, 4, 5, 6, 7,8, 9, and 10, or any range or subset of those values, and fractionalvalues when appropriate. Similarly, ranges provided as “up to” a certainvalue are understood to include values from zero to the top end of therange; and “less than” is understood to include values from that numberto zero, e.g., less than 5 is understood as 5, 4, 3, 2, 1, or 0, or afractional portion of a value within the range of 5 to 0. “One or morethan” is understood to include one and all values greater than 1, e.g.,1, 2, 3, 4, 5, 6, 7, 8, etc. or any specific value starting with 1.

“At least a portion” is understood as some fraction (e.g., at leastabout 1%) of the time, volume, or other entity from which the portion isobtained, to essentially all of (e.g., about 100%) or all of. Forexample, a sample mixed or heated during an incubation period need notbe mixed or heated throughout the entire incubation period. Similarly,typically less than all of the second extraction buffer is analyzed todetermine the concentration of CoQ10 in the extracted from the sample.

When ratios of liquids or percent solutions/suspensions of liquids areprovided, unless stated otherwise, the ratios are volume to volume. Whenpercent solutions of solids are provided, unless state otherwise, theratios are weight to volume.

The recitation of a listing of chemical group(s) in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof. When no stereochemistry is provided, all stereoisomers, racemicmixtures, or single stereoisomers are included in the term. When both astructure and the name of the structure are provided, in the event of adiscrepancy, the structure predominates over the name.

Any embodiment, method, or kit provided herein can be combined with anyother embodiment, method, or kit provided herein.

II. Methods and Uses

Extraction Methods

Provided herein are method and kits for the determination of the amountof CoQ 10 in a biological sample. The extraction methods provided hereincan also be used with non-spectrophotometric detection methods,including methods in which the CoQ10 is separated from other componentsin the sample based on size prior to detection.

The invention provides rapid, efficient methods for the detection ofCoQ10 in samples. The methods provided herein can be adapted forautomated high throughput screening methods, e.g., for use in clinicallaboratories. This is made possible by both the high level of extractionof CoQ10 obtained using the methods provided herein, and by detection ofCoQ10 using spectroscopy methods that do not require the isolation ofCoQ10 or the detection of CoQ10 based on molecular weight, which allowsthe method to be adaptable for high throughput screening methods. Theextraction methods provided herein can also be used with methods ofdetection of CoQ10 that include separation of CoQ10 by size, e.g.,liquid chromatography, prior to detection. In some embodiments, liquidchromatography is following by mass spectrometric analysis of CoQ10present in the sample. However, preferred methods of the invention donot include a step for separation of CoQ10 based on the size from othercomponents present in the sample.

In certain embodiments, the methods provided include adding a firstextraction buffer and a second extraction buffer to a sample, e.g., atissue sample or cell sample. The phases then are allowed to separate,and the CoQ10 is retained in the second extraction buffer layer. Thesecond extraction layer is then analyzed spectroscopically to determinethe amount of CoQ10 present in the sample.

In certain embodiments, the first and second extraction buffers areadded to the sample at the same time (e.g., as an emulsion; sequentiallywith no predefined incubation time between additions, e.g., within 30seconds of each other, within 20 seconds of each other, within 10seconds of each other; with no defined steps, e.g., mixing and/orheating in between). In certain embodiments, the first and secondextraction buffers are added to the sample prior to actively mixing thecomponents together, e.g., by mechanically mixing, sonication, or usinga magnetic stirrer. In certain embodiments, the sample is incubated withthe first extraction buffer prior to addition of the second extractionbuffer. In certain embodiments, a surfactant or detergent is added tothe sample. In certain embodiments, the surfactant or detergent is mixedwith the first extraction buffer prior to addition of the firstextraction buffer to the sample. In certain embodiments, the surfactantor detergent is added to the sample at the same time as the firstextraction buffer, and optionally at the same time as the secondextraction buffer. In certain embodiments, an inorganic salt is added tothe sample after the addition of the second extraction buffer. Incertain embodiments, the inorganic salt is a chloride salt such assodium chloride. In certain embodiments, the salt is added as asaturated salt solution, preferably in water. In certain embodiments,the salt would be present at a final concentration of about 1 mM toabout 50 mM (e.g., about 5 mM to about 45 mM, about 10 mM to about 35mM, about 1 mM to about 10 mM, about 1 mM to about 25 mM, about 10 mM toabout 50 mM, about 25 mM to about 50 mM, about 20 mM to about 30 mM;about 15 mM to about 35 mM; or any range bracketed by the valuesprovided).

In certain embodiments, the sample is heated during at least a portionof the incubation period with the first extraction buffer, andoptionally with the surfactant or detergent, during at least a portionof the incubation period. In certain embodiments, the sample is mixedduring at least a portion of the incubation period. In certainembodiments, the sample is mixed and/or heated after the addition of thesecond extraction buffer. In certain embodiments, the sample isincubated after the addition of both the first and second extractionbuffers, and optionally the surfactant or detergent. In certainembodiments, the sample is heated in the presence of the firstextraction buffer. In certain embodiments, the sample is heated in thepresence of the second extraction buffer. In certain embodiments, thesample is heated in the presence of the first and second extractionbuffers. In certain embodiments, the sample is heated in the firstbuffer, or the second buffer, or in both the first and second buffersfor at least 5 seconds, at least 10 seconds, at least 15 seconds, atleast 20 seconds, at least 30 seconds, at least 40 seconds, at least 50seconds, at least 60 seconds, at least 75 seconds, at least 90 seconds,at least 105 seconds, or at least 120 seconds. In certain embodiments,the heating step increases the amount of CoQ10 extracted as compared toa sample not heated during extraction by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, or at least 50%, ormore.

The differences in the physical properties of the first extractionbuffer and the second extraction buffer results in spontaneous phaseseparation of the first and second extraction buffers. In certainembodiments, partitioning can be promoted by cooling a heated sample,either actively or passively, to ambient temperature (i.e., standardlaboratory temperature, temperature inside an automated, high throughputassay apparatus). Upon partitioning of the phases, the CoQ10 is presentin the second extraction phase, i.e., in the non-polar solvent.

In certain embodiments, after partitioning, at least a portion of thesecond extraction phase is removed from the sample to facilitatespectroscopic analysis. In certain embodiments, the second extractionphase is removed to a quartz container (e.g., a cuvette, anappropriately shaped tube) for reading at a wavelength of 275 nm toquantitatively detect the presence of CoQ10. The invention provides anadvantage over other quantitative methods for the detection of CoQ10which require the isolation of CoQ10 from substantially all of the othercomponents of the sample to allow for its detection based on size, e.g.,using chromatographic methods or mass spectrometry methods which requirethe separation of molecular species within a mixture, e.g., byseparation on a column or electrospray, followed by detection andidentification of various components of the species within the mixture.The extraction method provided herein allows for the quantitativedetection of CoQ10 in a sample without detecting CoQ10 based onmolecular weight. As result, the method is readily adaptable to highthroughput methods and can be preformed quickly (e.g., in about 10minutes or less; in about 5 minutes or less; in about 2 minutes or less;in about 1 minutes or less; in about 30 seconds or less).

The extraction methods provided herein are highly efficient atextracting CoQ10 from sample material, e.g., typically biologicalmaterial such as cells either grown in culture or from subject samples.Extraction efficiency is determined as the amount of CoQ10 extractedusing the methods of the invention relative to the amount of CoQ10extracted using methanol alone, the method routinely used for CoQ10extraction prior to detection using liquid chromatograph followed bymass spectrophotometric methods. Extraction efficiency is expressed asthe fold increase in efficiency over extraction methods using onlymethanol, and preferably extraction methods using only methanol followedby liquid chromatography separation, and mass spectrometry detectionmethods. Using the methods of the invention, extraction is at least, 2-,3-, 4-, 5-, 7-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 75-, or 100-fold ormore efficient. In certain embodiments, extraction is at least or about5-, 11-, 14-, 28-, 82-, 545-, or 1863-fold more efficient.

The amount of sample used and the volume of the extraction depend on anumber of factors including, but not limited to, the amount of sampleavailable, the volume of the extraction vial and the cuvette for thespectrophotometric analysis. For example, when samples are processedmanually, larger volumes are typically used, whereas when automatedmethods are used, smaller sample volumes may be used.

The ratio of the first extraction buffer to the second extraction bufferis preferably about 5:1 to about 1:1 (v/v) (e.g., 5:1, 4:1, 3:1, 2:1,1:1, or fractional values within the range bracketed by any of thevalues, e.g., 4.5:1, 3.3:1, 2.1:1). The ratio of the surfactant ordetergent, when present, to the first extraction buffer is preferablyabout 1:50 to about 1:1 (v/v) (1:50, 1:45. 1:40, 1:35, 1:30, 1:25, 1:20,1:15; 1:10, 1:5, 1:1). The concentration of the surfactant or detergentis preferably about 0.01 mM to about 10 mM (e.g., about 0.01 mM to about1 mM, about 1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 0.01mM to about 2 mM, about 0.1 mM to about 7.5 mM, about 1 mM to about 10mM, or any range bracketed by any of the values provided). It isunderstood that some detergents and surfactants are mixed polymershaving approximate molecular weights, and therefore only approximatemolarities can be provided.

Use of CoQ10-d6 and CoQ10H₂-d6 as Internal Standards

Also provided herein are methods of detection and quantification ofoxidized and reduced forms of CoQ10 in a sample using mass spectrometry,e.g., LC-MS/MS. The oxidized and reduced forms of CoQ10 in a sample maybe detected and quantified separately or they both may be detected andquantified together, in a single analytical run. In a specificembodiment, the methods of the present invention may be used to quantifythe amount of reduced CoQ10 (CoQ10H₂) present in a sample. These methodsallow accurate and reliable quantification of CoQ10H₂, which isimportant because CoQ10H₂ is the biologically active form of CoQ10.

In some embodiments, the methods of detection and quantification ofoxidized and reduced forms of CoQ10 may be combined with the sampleextraction methods described herein or with other extraction methods.For example, in some embodiments, the reduced and oxidized forms ofCoQ10 in a sample, e.g., a biological sample, may be detected andquantified by extracting the sample using one extraction buffer, e.g.,1-propanol. In other embodiments, the reduced and oxidized forms ofCoQ10 in a sample, e.g., a biological sample, may be detected andquantified by extracting the sample using a first and a secondextraction buffer according to the methods described herein.

Quantification of the oxidized and reduced forms of CoQ10 isaccomplished by using their respective isotopically labeled versions asinternal standards, e.g., oxidized and reduced deuterated COQ10. In someembodiments, CoQ10-d6 is used as an internal standard for determiningthe amount of CoQ10 in a sample. In some embodiments, CoQ10H₂-d6 is usedas an internal standard for determining the amount of CoQ10H₂ in asample. In some embodiments, CoQ10-d6 and CoQ10H₂-d6 are both added tothe sample to be used for simultaneous determination of the amounts ofCoQ10 and CoQ10H₂ contained in the sample. The amount of CoQ10 in asample can be calculated based on the known amount of CoQ10-d6 added tothe sample and on the relative mass spectrometric signals produced byCoQ10 and CoQ10-d6. Similarly, the amount of CoQ10H₂ in a sample can becalculated based on the known amount of CoQ10H₂-d6 added to the sampleand on the relative mass spectrometric signals produced by CoQ10H₂ andCoQ10H₂-d6.

The use of CoQ10-d6 as an internal standard for quantifying CoQ10 in asample, and the use of CoQ10H₂-d6 for quantifying CoQ10H₂ in a sampleprovides advantages over the known methods that employ CoQ10-d6 oranalogs such as CoQ9 or CoQ11, as internal standards for quantifyingboth CoQ10 and CoQ10H₂. Specifically, the quantification methodsdescribed herein provide greater accuracy of CoQ10H₂ quantificationbecause CoQ10H₂-d6, used in the methods as an internal standard, hasproperties, e.g., extraction recovery, ionization response andchromatographic retention time that are very similar to those ofCoQ10H₂, but also produces different mass spectrometric signals. Incontrast, the use of CoQ10-d6, CoQ9 or CoQ11 as internal standards forquantifying CoQ10H₂ results in a biased quantification and loweraccuracy.

The internal standard, e.g., CoQ10-d6 or CoQ10H₂-d6, can be added to asample at any point of the sample work-up procedure. In someembodiments, the internal standard is added to each sample in thebeginning of sample processing, e.g., extraction. In some embodiments,the sample comprises blood, and the internal standard is added to thesample prior to plasma extraction.

The amount of internal standard, e.g., CoQ10-d6 or CoQ10H₂-d6, to beadded to each sample should not interfere with the mass spectrometricsignal produced by the analyte, CoQ10 or CoQ10H₂. In some embodiments,the amount of CoQ10-d6 or CoQ10H₂-d6 to be added to each sample shouldresult in a concentration of CoQ10-d6 or CoQ10H₂-d6 that is within thelinear range of the dose response curve. In a preferred embodiment, theamount of CoQ10-d6 or CoQ10H₂-d6 to be added to each sample shouldproduce a concentration between 20 and 2500 ng/mL, (e.g., 50 and 2000ng/ml, or 100 and 1500 ng/ml or between 100 and 1000 ng/ml).

In some embodiments, the present invention provides methods fordetermining the extent of CoQ10H₂ degradation that occurs during and asa result of sample processing, e.g., sample extraction. An example ofCoQ10H₂ degradation is oxidation of CoQ10H₂ to produce a partiallyoxidized form (ubisemiquinone, CoQ10H) or fully oxidized form(ubiquinone, CoQ10). Degradation of CoQ10H₂ can be exacerbated byexposure to light and high temperatures that occurs during sampleprocessing and before the sample is introduced into mass spectrometerfor detection of CoQ10 and/or CoQ10H₂. In some embodiments, the extentof CoQ10H₂ oxidation can be calculated based on the known amount ofCoQ10H₂-d6 added to the sample prior to sample processing and on themeasured relative signals of the remaining CoQ10H₂-d6 and of CoQ10H-d6and CoQ10-d6 resulting from oxidation. In a preferred embodiment, themeasured amounts of CoQ10 and CoQ10H₂ in a sample are adjusted by theamount of CoQ10H₂-d6 that becomes oxidized and the amount of CoQ10-d6that is produced during sample processing.

Methods of CoQ10 and CoQ10H2 Detection Using Mass Spectrometry andInternal Standards

In certain embodiments, the invention provides an LC-MS/MS method fordetermining the amount of CoQ10 and CoQ10H₂. This method is linear overthe clinically relevant range of 20-2500 ng/mL for both forms, withr²>0.99. The % CV's and inter and intra-assay precision/accuracy valuesfor both forms is below 10%.

In some embodiments, the invention provides methods of determining theamount of CoQ10 and/or CoQ10H₂ in a sample, the method comprising:

a) providing the sample;

b) optionally adding a known amount of CoQ10-d6 and/or CoQ10H₂-d6 to thesample;

c) processing the sample;

d) optionally subjecting the sample to liquid chromatography;

e) detecting CoQ10 and/or CoQ10H₂ and, if applicable, CoQ10-d6 andCoQ10H₂-d6 by mass spectrometry; and

f) determining the amount of detected CoQ10 and/or CoQ10H₂ in the sampleby comparing it to the known amount of detected CoQ10H₂-d6.

In some embodiments, the entire method is carried out in reduced lightand using amber vials to minimize degradation of CoQ10H₂. In someembodiments, the sample collection is carried out using pre-cooledcollection tubes to minimize temperature-induced degradation of CoQ10H₂.In a further embodiment, the collection tubes may be pre-cooled to atemperature between 0° C. and −20° C. In the preferred embodiment, thecollection tubes may be pre-cooled to −20° C. In another embodiment, thecollection tubes may be pre-cooled to a temperature of between about−20° C. and -about 80° C., e.g, about −20° C., about −25° C., about −30°C., about −35° C., about −40° C., about −45° C., about −50° C., about−55° C., about −60° C., about −65° C., about −70° C., about −75° C., orabout −80° C.

In some embodiments, the sample is blood, e.g., human blood that iscollected in pre-cooled BD Vacutainer® Tubes containing Lithium Heparinas the anti-coagulant and processed immediately using standardprocedures known in the art that can be used to extract plasma fromblood. In a further embodiment, the plasma is separated within 30,preferably, 15 minutes of blood sample collection. In an alternativeembodiment, the sample is blood, e.g., human blood that is collected inpre-cooled heparinized vials and kept at low temperature.

In some embodiments, where the blood sample has not been processedwithin 30 minutes of collection to extract plasma, step c may comprisethe extraction of plasma using any plasma extraction method known in theart.

In some embodiments, step c may comprise protein precipitation to removemost of the protein from the sample, leaving CoQ10, CoQ10H₂ and theirisotopically labeled analogs, if applicable, in the supernatant. Thesamples may be centrifuged to separate the liquid supernatant from theprecipitated proteins; alternatively the samples may be filtered toremove precipitated proteins. The resultant supernatant or filtrate maythen be applied directly to mass spectrometry analysis; or alternativelyto liquid chromatography and subsequent mass spectrometry analysis.

In other embodiments, step c may comprise protein precipitation usingextraction with a single solvent. In some embodiments, the solvent forextraction is selected, such that the analyte molecules, e.g., CoQ10 andCoQ10H₂, are stable, e.g., not degraded to a significant extent, over aperiod of time, when placed in the solvent. In some embodiments, theperiod of time can be up to 6 hours, e.g., 5 minutes, 10 minutes, 30minutes, 60 minutes, 2 hours, 4 hours or 6 hours. In some embodiments,the solvent may be hexane, methanol, or 1-propanol. In some embodiments,step c is carried out using pre-cooled test tubes, pre-cooled cryo-blockand pre-cooled solvents to minimize temperature-induced degradation ofCoQ10H₂. In a further embodiment, the test tubes, the cryo-block and thesolvents may be pre-cooled to a temperature between about 0° C. andabout −20° C., e.g., about −2° C. about −5° C., about −10° C., about−12° C., about −15° C., or about −20° C. In the preferred embodiment,the test tubes, the cryo-block and the solvents may be pre-cooled to atemperature of about −20° C. In another embodiment, the test tubes, thecryo-block and the solvents may be pre-cooled to a temperature ofbetween about −20° C. and -about 80° C., .e.g, about −20° C., about −25°C., about −30° C., about −35° C., about −40° C., about −45° C., about−50° C., about −55° C., about −60° C., about −65° C., about −70° C.,about −75° C., or about −80° C.

In some embodiments, step c may comprise sample extraction using a firstextraction buffer and a second extraction buffer, as is describedelsewhere in this application.

In some embodiments, the extracted sample may be further subjected toliquid chromatography in step d to separate CoQ10 and CoQ10H₂ and theirisotopically labeled analogs, if applicable. Traditional chromatographicanalysis relies on column packing in which laminar flow of the samplethrough the column is the basis for separation of the analyte ofinterest from the sample. The skilled artisan will be able to select LC,including HPLC, instruments and columns that are suitable for use withCoQ10 and CoQ10H₂. The chromatographic column typically includes amedium (i.e., a packing material) to facilitate separation of chemicalmoieties (i.e., fractionation). The medium may include minute particles,or may include a monolithic material with porous channels. A surface ofthe medium typically includes a bonded surface that interacts with thevarious chemical moieties to facilitate separation of the chemicalmoieties. One suitable bonded surface is a hydrophobic bonded surfacesuch as an alkyl bonded, cyano bonded surface, or highly pure silicasurface. Alkyl bonded surfaces may include C-4, C-8, C-12, or C-18bonded alkyl groups. In preferred embodiments, the column is a C18microparticle packed column (such as Agilent C18 Zorbax column). Thechromatographic column includes an inlet port for receiving a sample andan outlet port for discharging an effluent that includes thefractionated sample.

Any chromatographic method that results in effective separation of CoQ10and CoQ10H₂ may be used. In some embodiments, the elution of CoQ10 andCoQ10H₂ from a reversed phase column is accomplished using an isocraticelution mode, i.e., wherein the composition of the mobile phase is keptcontant. In some embodiments, the composition of mobile phase is 30:70to 90:10 A:B, wherein A is 5 mM ammonium formate and B is 1-propanol.The composition of the mobile phase can be changed by substituting 5 mMammonium formate and 1-propanol with any other solvents having similarpolarity, including, but not limited to ethanol, 2-propanol, acetone oracetonitrile. In a preferred embodiment, the composition of the mobilephase is 80:20 A:B, wherein A is 5 mM ammonium formate and B is1-propanol, and the chromatographic separation is carried out for 5minutes.

In some embodiments, CoQ10 and CoQ10H₂ and their isotopically labeledanalogs, if applicable, may be detected during chromatography. In someembodiments, the detection may comprise spectroscopic detection. CoQ10is preferably detected at a wavelength near or at 275 nm (e.g., 270-280nm; 272-278 nm; 274-276 nm), using ultraviolet spectroscopy.

In preferred embodiments, the eluted CoQ10 and CoQ10H₂ and theirisotopically labeled analogs, if applicable, are fed directly into amass spectrometer after chromatographic separation. In an alternativeembodiments, the chromatographic fraction comprising the eluted CoQ10and CoQ10H₂ and their isotopically labeled analogs, if applicable, mayfirst be collected and then introduced into a mass spectrometer in aseparate step.

In step e, CoQ10 and CoQ10H₂ and their isotopically labeled analogs, ifapplicable, are detected using mass spectrometry. During massspectrometry, CoQ10 and CoQ10H₂ may be ionized by any method known tothe skilled artisan. Mass spectrometry is performed using a massspectrometer, which includes an ion source for ionizing the fractionatedsample and creating charged molecules for further analysis. For exampleionization of the sample may be performed by electron ionization,chemical ionization, electrospray ionization (ESI), photon ionization,atmospheric pressure chemical ionization (APCI), photoionization,atmospheric pressure photoionization (APPI), Laser diode thermaldesorption (LDTD), fast atom bombardment (FAB), liquid secondaryionization (LSI), matrix assisted laser desorption ionization (MALDI),field ionization, field desorption, thermospray/plasmaspray ionization,surface enhanced laser desorption ionization (SELDI), inductivelycoupled plasma (ICP) and particle beam ionization. The skilled artisanwill understand that the choice of ionization method may be determinedbased on the analyte to be measured, type of sample, the type ofdetector, the choice of positive versus negative mode, etc.

CoQ10 and CoQ10H₂ and their isotopically labeled analogs may be ionizedin positive or negative mode. In a preferred embodiment, CoQ10 andCoQ10H₂ and their isotopically labeled analogs are ionized using ESI inpositive ion mode.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass-to-charge ratio. Suitable analyzers fordetermining mass-to-charge ratios include quadrupole analyzers, iontraps analyzers, and time-of-flight analyzers. Exemplary ion trapmethods are described in Bartolucci, et al., Rapid Commun. MassSpectrom. 2000, 14:967-73.

The ions may be detected using several detection modes. For example,selected ions may be detected, i.e. using a selective ion monitoringmode (SIM), or alternatively, mass transitions resulting from collisioninduced dissociation or neutral loss may be monitored, e.g., multiplereaction monitoring (MRM) or selected reaction monitoring (SRM). In someembodiments, the mass-to-charge ratio is determined using a quadrupoleanalyzer. For example, in a “quadrupole” or “quadrupole ion trap”instrument, ions in an oscillating radio frequency field experience aforce proportional to the DC potential applied between electrodes, theamplitude of the RF signal, and the mass/charge ratio. The voltage andamplitude may be selected so that only ions having a particularmass/charge ratio travel the length of the quadrupole, while all otherions are deflected. Thus, quadrupole instruments may act as both a “massfilter” and as a “mass detector” for the ions injected into theinstrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions.

Alternate modes of operating a tandem mass spectrometric instrumentinclude product ion scanning and precursor ion scanning. For adescription of these modes of operation, see, e.g., E. Michael Thurman,et al., Chromatographic-Mass Spectrometric Food Analysis for TraceDetermination of Pesticide Residues, Chapter 8 (Amadeo R.Fernandez-Alba, ed., Elsevier 2005) (387).

The results of an analyte assay may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of vitamin Dmetabolites. Methods of generating and using such standard curves arewell known in the art and one of ordinary skill is capable of selectingan appropriate internal standard. For example, in preferred embodiments,CoQ10-d6 and CoQ10H₂-d6 may be used as internal standards. Numerousother methods for relating the amount of an ion to the amount of theoriginal molecule will be well known to those of ordinary skill in theart.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In some embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activated dissociation (CAD) isoften used to generate fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In some embodiments, CoQ10 and CoQ10H₂ in a sample are detected by massspectrometry using multiple reaction monitoring (MRM) as follows. Asample, e.g., a sample comprising chromatographic fractions of step dand a solvent, enters the nebulizer interface of an MS/MS analyzer andis converted to vapor in the heated charged tubing of the interface. Theanalyte(s) (CoQ10 and CoQ10H₂ and their isotopically labeled analogs, ifapplicable), contained in the sample, are ionized by applying a largevoltage to the solvent/analyte mixture. As the analytes exit the chargedtubing of the interface, the solvent/analyte mixture nebulizes and thesolvent evaporates, leaving analyte ions. The ions, e.g. precursor ions,pass through the orifice of the instrument and enter the firstquadrupole. Quadruples 1 and 3 (Q1 and Q3) are mass filters, allowingselection of ions (i.e., selection of “precursor” and “fragment” ions inQ1 and Q3, respectively) based on their mass to charge ratio (m/z).Quadrupole 2 (Q2) is the collision cell, where ions are fragmented. Thefirst quadrupole of the mass spectrometer (Q1) selects for moleculeswith the mass to charge ratios of derivatized vitamin D metabolites ofinterest. Precursor ions with the correct mass/charge ratios are allowedto pass into the collision chamber (Q2), while unwanted ions with anyother mass/charge ratio collide with the sides of the quadrupole and areeliminated. In some embodiments, the precursor product ion for CoQ10 maybe the ion of m/z 863.4, and the precursor product ion for CoQ10H₂ maybe the ion of m/z 865.4. Using standard methods well known in the art,one of ordinary skill is capable of identifying one or more fragmentions of a particular precursor ion of CoQ10, CoQ10H₂, CoQ10-d6 andCoQ10H₂-d6 that may be used for further fragmentation in quadrupole 2(Q2).

Precursor ions entering Q2 collide with neutral argon gas molecules andfragment. The fragment ions generated (i.e., product ions) are passedinto quadrupole 3 (Q3), where the fragment ions of analytes are selectedwhile other ions are eliminated. In some embodiments, the product ion ofm/z 197 are detected for both CoQ10 and CoQ10H₂.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of CoQ10, CoQ10H₂,CoQ10-d6 and CoQ10H₂-d6 that may be used for selection in quadrupole 3(Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theareas under the peaks corresponding to particular ions, or the amplitudeof such peaks, may be measured and correlated to the amount of theanalyte of interest. In certain embodiments, the area under the curves,or amplitude of the peaks, for fragment ion(s) and/or precursor ions aremeasured to determine the amount of CoQ10 or CoQ10H₂. As describedabove, the relative abundance of a given ion may be converted into anabsolute amount of the original analyte using calibration standardcurves based on peaks of one or more ions of an internal standard, e.g.,CoQ10-d6 or CoQ10H₂-d6.

The following examples are illustrative of the methods of the inventionand should not be understood to limit the scope of the invention.

EXAMPLES Example 1—Materials and Methods

Cell Culture

The assay for quantitation of CoQ10 was used to determine the CoQ10levels in various cells lines after CoQ10 treatment for various times.The cell lines used in the studies were the normal human aortic smoothmuscle cells (HASMC), the hepatic cancer cell line HepG2, and the humanpancreatic carcinoma cell line PaCa2. All cells lines are commerciallyavailable and were maintained according to instruction from themanufacturer/supplier of the cell lines.

Preparing CoQ10 Standard Solution

A 500 μM CoQ10 stock solution was prepared by weighing 4.32 mg of CoQ10in a 15 ml conical tube, adding 10 ml of hexane, warming the tube in awater bath at 65° C. until the CoQ10 dissolved, and shaking the tubegently to mix the solution. A 100 μM and 50 μM CoQ10 stock solutionswere prepared by diluting the stock solution in hexane.

Reagent Compositions

Buffer A (First extraction buffer): 100% Methanol

Buffer B (Surfactant/detergent): 1 mM Na-deoxycholate

Buffer C (Second extraction Buffer): 100% Hexane

General Methods: Extraction/Analysis of a Standard Solution

Step 1:

A 500 μM stock solution of CoQ10 was prepared in hexane as describedabove and used to prepare 100 μM and 50 μM solutions of CoQ10 in theappropriate buffer for cell treatment or for direct use in the detectionassay methods.

Step 2:

200 μl of various concentrations of CoQ10 in Buffer A, or Buffer Aalone, were aliquoted into glass vials and 10 μl of Buffer B was addedto each vial. Cells treated with various concentrations of CoQ10 werecollected at predetermined time points and washed. Cell pellet samples(e.g., about 10⁵-10⁷ cells) were combined with 200 μl of Buffer A and 10μl of Buffer B, and the cell pellets were resuspended in the buffermixture. The sample mixture turned opaque upon mixing Buffers A and Bwith cells. The mixtures were transferred into glass vials.

Step 3:

The glass vials from step 2 were warmed to 65° C. for 1 minute. Thesamples turned clear upon heating, and again became opaque upon coolingto room temperature.

Step 4:

A 200 μl aliquot of Buffer C was added to each vial. The vials wereagain warmed to 65° C. The solution in the vials spontaneouslypartitioned into two phases, with the second extraction buffer layer(upper layer) containing substantially all of the CoQ10 material.

Step 5:

A 50 μl sample of the upper extraction layer was removed and analyzed ona UV spectrometer at 275 nm. The amount of CoQ10 in each sample wasdetermined by comparison to a standard curve.

Example 2—Generation of Standard Curves for CoQ10 Concentrations inVarious Solvents

Various solvent systems were tested to optimize the extraction process.To determine optimal spectral properties of the solvent system duringanalysis using UV spectroscopic detection of CoQ10, standard curves weregenerated by spectroscopy. Three different solvents (2-propanol,acetonitrile, and hexane) were tested, using known concentrations ofCoQ10.

To prepare a standard curve, a 50 μM CoQ10 stock was prepared in theappropriate solvent and serial dilutions of the stock were made. TheOD₂₇₅ of each of the samples from the serial dilution was measured at275 nm in a UV spectrophotometer and plotted on a graph. The results ofthe standard curve preparation are presented in Table 2 and FIG. 1.

TABLE 2 OD₂₇₅ values for various concentrations of CoQ10 in differentsolvents Concentration of CoQ10 (μM) Hexane 2-Propanol Acetonitrile 500.652 0.614 0.617 25 0.344 0.3 0.304 12.5 0.166 0.154 0.145 6.25 0.0690.075 0.065 3.13 0.034 0.035 0.03 1.56 0.016 0.016 0.012 0.781 0.0070.006 0.006 0.391 0.001 0.001 0

A linear regression analysis of the data was performed to generate thestandard curves. These results revealed that the spectra of the2-propanol and acetonitrile solutions were identical, and that thehexane solution deviated slightly in the upper portion of the graph. Allthree solvents provided results that could be fitted to a linearequation over a substantial range of concentrations Any of the threestandard curves could be used to determine the concentration of CoQ10 inthe appropriate solvent based on the OD₂₇₅.

Example 3—Quantitation of CoQ10 in Tissue Culture Cells Exposed toVarying Concentrations of CoQ10

Extraction of CoQ10 from Cell Samples

In the experimental trials, about 1×10⁶ HASMC, HepG2, or PaCa2 cellswere plated into each well of a 6 well plate. At the time of plating,the cells were treated with defined concentrations of CoQ10, intriplicate, in isopropanol or a proprietary CoQ10 delivery formulationfor 3, 6, or 24 hours. After the defined incubation time, the cells werewashed with cold TPBS (PBS with 100 mM Tris, pH 7.4), harvested bytrypsinization, washed (2×) with 1 ml of TPBS, and pelleted bycentrifugation at 1,500 RPM. Immediately after trypsinization, the cellswere counted to determine the total number of viable cells in thesample.

Cell pellets were resuspended in 200 μL of Buffer A (first extractionbuffer) and 10 μL of Buffer B (surfactant/detergent). Upon addition ofBuffer B, the sample became opaque and a precipitate formed. Theresuspended cells were transferred to a borosilicate vial. For controlsamples not containing cells, the CoQ10 containing solution was added toa glass vial and hexane was added to bring the volume to 200 μl, and 10μl of Buffer B was then added.

The vials were incubated at 65° C. for 2 minutes in a water bath, withgentle mixing at about half way through the incubation. The samplechanged from cloudy to clear during the incubation. The sample wasremoved from the water bath and 200 μL of Buffer C (second extractionbuffer) was added to the vial. The sample partitioned into two phases,polar and non-polar. The top, non-polar, layer was clear, while a whiteprecipitate formed the bottom of the vial. The sample was again heatedat 65° C. for 1 minute with gentle mixing about half way through theincubation. The solution was cooled to room temperature and a 75 μLaliquot of the upper phase was placed in a quartz cuvette and analyzedby UV spectrophotometry at a wavelength of 275 nm. The absorption wasdocumented.

Generation of Standard Curve

For quantitative assessment of CoQ10 in the biological samples, astandard curve was generated essentially as set forth above (Example 2).For each independent determination of CoQ10, the standard curve wasgenerated by making a 50 μM CoQ10 stock solution in hexane and seriallydiluting of the stock material. Each of the samples from the serialdilution was measured at a wavelength of 275 nm in a UV spectrometer andplotted on a graph. A representative standard curve is presented in FIG.2.

A power series was used to fit the standard curve from the standard andan equation was determined. The curve did not fit a typical linearregression form. This lack of linearity demonstrates the benefit ofgenerating a standard curve for each experiment, and the importance ofpreferably calculating CoQ10 concentrations in unknown samples fromOD₂₇₅ readings falling within the linear portion of the curve. Using theOD₂₇₅ values from the extracted samples, the amount of CoQ10 wasdetermined by them against the generated standard curves.

Data Analysis

For this study all cell samples were repeated in triplicate. CoQ10extractions and OD₂₇₅ measurements were performed by individuals notinvolved in CoQ10 treatment of the cells or harvesting. As an initialbenchmark of the methods used, the data were analyzed forreproducibility.

Hep2G cells not treated with CoQ10, but simply harvested at theindicated times after plating, provided highly reproducible results forall replicate samples (see FIG. 3A). This demonstrates thereproducibility of the extraction and detection methods.

In regard to reproducibility, more variation was seen in cells collectedafter treatment with various concentrations of CoQ10 for 3, 6, and 24hours within the triplicate wells. As shown in FIGS. 3B-E, variation inthe amount of CoQ10 in Hep 2G cells was observed most substantially atthe 24 hour time point.

The results were also analyzed for the efficiency of CoQ10 uptake intocells depending on the delivery vehicle. These results are summarized inTable 3 below. As evident from the data, substantially more CoQ10 waspresent in cells after 24 when the CoQ10 was delivered in the CoQ10proprietary delivery formulation than in propanol. The amount of CoQ10is calculated based on pM of CoQ10 per cell; therefore, the observeddifference in intracellular CoQ10 is not a result of a difference incell viability in response to the two carriers or time to proliferate inculture. Despite the nominal statistical deviations, the data obtainedby using the method provided herein were shown to be statisticallyreliable and the method is reproducible.

TABLE 3 Summary of average levels of CoQ10 in Hep2G cells treated withCoQ10 (pM/cell) 3 hours 6 hours 24 hours Untreated 14.63 27.40 38.07 50μM in propanol 14.40 27.07 37.04 100 μM in propanol 25.20 34.74 45.43 50μM in delivery 28.1 30.2 227.57 formulation 100 μM in delivery 24.7 30.2717 formulation

Example 4—Comparison of the In Vitro Spectrophotometric Method VersusLC/MS/MS (MRM) Method for CoQ10 Quantitation in Biological Samples

To further validate the utility of the spectrophotometric method for thequantitative assessment of CoQ10 in biological samples, the experimentdescribed above was repeated using HASMC, HepG2, and PaCa2 cell lines.Further, a second set of cell samples were prepared using each of thethree cell types for analysis to determine CoQ10 levels using LC/MS/MSmethods routinely used in the art. Briefly, cells were plated andtreated with CoQ10 in isopropanol or the CoQ10 proprietary deliveryformulation as set forth above, and harvested at 3, 6, or 24 hours afterplating. Cell pellets were either extracted using the method set forthabove, resuspending the cells in Buffers A and B, and extracting theCoQ10 with Buffer C, or the cell pellets were sent to a diagnosticlaboratory (IriSys Research & Development Inc) for quantitativeassessment of CoQ10 using extraction with methanol alone followed byLC/MS/MS detection.

The data from the two analyses are summarized in Table 4 below. It isapparent that extraction of CoQ10 from biological samples using methanolalone followed by LC/MS/MS detection resulted in an underestimation ofCoQ10 levels in every sample tested and under all treatment conditionswhen compared to the extraction method described herein followed byspectrophotometric detection. These results demonstrate the efficacy ofthe extraction and quantitation methods for CoQ10 in biological samplesprovided herein as compared to techniques that are currently in use.These results also demonstrate more efficient delivery of CoQ10 to cellsusing the proprietary CoQ10 delivery formulation as compared toisopropanol.

TABLE 4 Comparison of Spectrophotometric and LC/MS/MS Detection of CoQ10HepG2 Cells Sample CoQ10 Cells/ Conc (pM/ # Treatment ReplicateFormulation ml (×10⁵) OD₂₇₅ CoQ10[μM] cell) 1 Untreated 1 Isopropanol10.2 0.187 29.99 29.4 2 Untreated 2 Isopropanol 7.5 0.201 32.23 42.9 3Untreated 3 Isopropanol 8.03 0.21  33.67 41.9 4 Untreated 1 Isopropanol6.2 LC/MS/MS 0.0873 0.014 5 Untreated 2 Isopropanol 7.1 LC/MS/MS 0.07170.010 6 Untreated 3 Isopropanol 8.0 LC/MS/MS 0.0788 0.010 7  50 μM 1Isopropanol 13.4 0.248 39.76 29.6 8  50 μM 2 Isopropanol 13.6 0.26141.84 30.7 9  50 μM 3 Isopropanol 9.9 0.321 51.45 51.9 10 100 μM 1Isopropanol 5.1 0.225 36.08 70.7 11 100 μM 2 Isopropanol 4.1 0.31  49.71121.2 12 100 μM 3 Isopropanol 5.0 0.315 50.51 101 13 100 μM 1Isopropanol 6.1 LC/MS/MS 2.061 0.34 14 100 μM 2 Isopropanol 5.1 LC/MS/MS1.223 0.24 15 100 μM 3 Isopropanol 6.6 LC/MS/MS 1.599 0.24 16 100 μM 1CoQ10 delivery 6.8 53 779 formulation 17 100 μM 2 CoQ10 delivery 8.365.2 785 formulation 18 100 μM 3 CoQ10 delivery 11.8 69.27 587formulation 19 100 μM 1 CoQ10 delivery 7.2 LC/MS/MS 5.948 0.83formulation 20 100 μM 2 CoQ10 delivery 7.7 LC/MS/MS 8.864 1.15formulation 21 100 μM 3 CoQ10 delivery 1.1 LC/MS/MS 6.099 0.18formulation Sample Cells/ Conc (pM/ # Treatment Replicate Formulation ml(×10⁵) OD₂₇₅ CoQ10[μM] cell) HASMC Cells 22 Untreated 1 Isopropanol 9.50.173 20.46 46.26 23 Untreated 2 Isopropanol 1.1 0.09  10.64 95.98 24Untreated 3 Isopropanol 0.3 0.051 6.02 187.87 25 Untreated 1 Isopropanol4.84 LC/MS/MS 0.0074 0.0015 26 Untreated 2 Isopropanol 7.92 LC/MS/MS0.1830 0.023 27 Untreated 3 Isopropanol 11.4 LC/MS/MS 0.0150 0.0013 28Untreated 2 Isopropanol 7.7 LC/MS/MS 0.0340 0.0044 29 Untreated 3Isopropanol 13.5 LC/MS/MS 0.0411 0.003 PaCa2 Cells 30 Untreated 1Isopropanol 0.61 0.057 6.79 5.51 31 Untreated 2 Isopropanol 0.20 0.0242.86 1.84 32 Untreated 3 Isopropanol 1.1 0.12  14.29 9.62 33 Untreated 1Isopropanol 15.0 LC/MS/MS 0.021 0.0014 34 Untreated 2 Isopropanol 14.0LC/MS/MS 0.045 0.0032 35 Untreated 3 Isopropanol 14.0 LC/MS/MS 0.230.016 36 100 μM 1 Isopropanol 13.2 0.239 28.46 1.56 37 100 μM 2Isopropanol 13.7 0.105 12.5 9.09 38 100 μM 3 Isopropanol 12.6 0.034 4.053.2 39 100 μM 1 Isopropanol 12.0 LC/MS/MS 0.371 0.31 40 100 μM 2Isopropanol 11.0 LC/MS/MS 1.917 0.17 41 100 μM 3 Isopropanol 9.9LC/MS/MS 1.186 0.12 42 100 μM 1 CoQ10 delivery 22.0 0.17  89.23 40.56formulation 43 100 μM 2 CoQ10 delivery 23.2 0.259 135.94 58.57formulation 44 100 μM 3 CoQ10 delivery 22.7 0.326 171.1 75.14formulation 45 100 μM 1 CoQ10 delivery 12.0 LC/MS/MS 1.501 0.12formulation 46 100 μM 2 CoQ10 delivery 11.0 LC/MS/MS 4.818 0.44formulation 47 100 μM 3 CoQ10 delivery 9.9 LC/MS/MS 7.26 0.73formulation

To facilitate comparison of the amount of CoQ10 detected in the cells,the fold difference of the concentration of CoQ10 per cell detectedusing the extraction and spectrophotometric methods provided herein wascompared to the amount of CoQ10 detected per cell using extraction withmethanol only followed by LC/MS/MS detection methods. The results areprovided in Table 5 below.

TABLE 5 Fold Difference in CoQ10 Detected in Cells using OD₂₇₅ vs.LC/MS/MS Treatment Cell Type Formulation Fold difference Untreated HepG2Isopropanol 28 100 μM HepG2 Isopropanol 82 100 μM HepG2 CoQ10 delivery 5formulation Untreated HASMC Isopropanol 545 Untreated PaCa2 Isopropanol1863 100 μM PaCa2 Isopropanol 11 100 μM PaCa2 CoQ10 delivery 14formulaton

It can be readily observed that the extraction and detection methodsprovided herein detect substantially more CoQ10 in a biological samplethan the LC/MS/MS methods routinely used in the art.

Example 5—Determination of Mass Spectrometric Parameters for Detectionof CoQ10 and CoQ10H₂

Oxidized CoQ10 Analysis

Methods:

5.04 mg of CoQ10 was dissolved in 1 mL of acetone for the resultingCoQ10 concentration of 5 mg/mL. A 100 μL aliquot of this solution wasmixed with 900 μL of isopropyl alcohol for the resulting CoQ10concentration of ≈0.5 mg/mL. A 200 μL aliquot of this solution was mixedwith 10 μL of formic acid and injected directly into the massspectrometer. For mass spectrometric analysis, Q1 scan was performed inthe positive ESI mode.

Results:

Molecular ion peak for the oxidized form was observed at m/z 863.4. Apossible peak for the reduced form of Coenzyme Q₁₀ (CoQ10H₂) wasobserved at m/z 885.4. A ghost peak observed at 199.2 Da, which may becaused by previous injections into the mass spectrometer or accumulationof molecules along the internal surfaces.

Product ion scan in the range of 150-800 Da was performed for m/z 863.4and revealed a single major product ion peat at m/z 197.0.

The peak at m/z 865.4 possibly corresponds to the reduced form of CoQ10(CoQ10H₂). This peak was observed in the Q1 scan but was not as intenseas the 863.4 Da peak. A product ion scan performed on this peak revealedm/z 197.0 as the major product peak. This corresponds to the basic ringstructure of Coenzyme Q₁₀.

A precursor ion scan for m/z 197 was performed and gave 863.9 Da as themajor precursor peak. When the m/z range of 850-900 Da was zoomed, alarge peak for 863.9, a lower intensity peak for 864.7 Da and a very lowintensity peak for 865.8 were observed. These peaks correspond to thecompletely oxidized (CoQ10) partially reduced (CoQ10H) and completelyreduced (CoQ10H₂) forms of Coenzyme Q₁₀.

Reduced CoQ10 Analysis

Methods:

5.032 mg of CoQ10 was dissolved in 1 mL of hexane to give stock solutionof 5 mg/mL stock solution. An aliquot of 100 μL was added to 900 μL ofhexane to give the second stock solution at the concentration of 500μg/mL. 100 μL of the second stock solution was diluted with 1.9 mL ofhexane, and then mixed with 50 μL of methanol and 20 mg of sodiumborohydride. The reduction reaction was stirred for 3 min and allowed tostand at room temperature in the dark for 5 min. Subsequently, 1 mLwater containing 100 mM EDTA was added to the reaction. The finalconcentration of CoQ10H₂ was 25 μg/mL. For mass spectrometric analysis,Q1 scan was performed in the positive ESI mode.

Results:

The Q1 scan for the reduced form of CoQ₁₀ did not reveal any molecularion peak which was consistent and could be related to the structure ofthe molecule. None of the expected peaks were observed. The possiblereason for this may be the addition of EDTA in the last step of thereduction process. EDTA may attach to the molecular ion and forminconsistent adducts, causing irreproducible results.

To remedy inconsistent results caused by EDTA, the above reductionprocedure was repeated, but in the last step, 1 mL of water without EDTAwas added. The reaction was then mixed with 5 mM ammonium formate inmethanol and infused into the mass spectrometer.

Two sets of peaks were observed in this infusion, one set at 863.8,864.8 and 865.8 m/z and another set at 880.4, 881.3 and 882.4 m/z. Thesepeaks corresponded to the completely oxidized (CoQ10) partially reduced(CoQ10H) and completely reduced (CoQ10H₂) forms of Coenzyme Q₁₀.

There was also a peak set observed at 880.4, 881.4 and 882.4 m/z thatcorresponded to the ammoniated adducts of the molecular peaks at 863.8,864.8 and 865.8 m/z. The product ion scans for all the above peaksrevealed 197.0 as the major/only product ion.

The Precursor Ion scan for 197.0 showed peaks for both the above sets ofm/z's. The set of peaks formed due to the ammonia adducts showed higherintensity and were used as precursor ions for finalizing massspectrometer parameters.

Example 6—Determination of Chromatographic Parameters for Detection ofCoQ10 and CoQ10H₂

Methods:

1 mg of CoQ10 was dissolved in 1 mL of Hexane, and 100 μL of thissolution was added to 900 μL of hexane, resulting in a 100 μg/mL stocksolution.

For the reduction reaction, 100 μL of stock solution was diluted with1.9 mL of hexane and mixed with 50 μL of methanol and 20 mg of sodiumborohydride. The reaction was stirred for 3 min and stored at roomtemperature in in the dark for 5 min. Complete conversion of theoxidized to the reduced form was confirmed visually by the change ofreaction color from yellow to colorless. Subsequently, 1 mL of water wasadded and mixed. The reaction was centrifuged at 13,000 rpm for 2 min toseparate the layers. The top hexane layer containing CoQ10H₂ at theconcentration of 5 μg/mL was collected (CoQ10H₂ stock).

The above reaction was repeated in another tube without the addition ofsodium borohydride. The top heane layer containing CoQ10 at theconcentration of 5 μg/mL was collected (CoQ10 stock).

Liquid chromatography was performed using 10 mM ammonium formate inmethanol as mobile phase A, 1-propanol as mobile phase B and the C18Zorbax column. The stocks containing CoQ10 and CoQ10H₂ were mixed at the1:1 ratio and injected into the chromatograph. Three isocratic 5 minuteruns were performed using the mobile phase where A and B were mixed atthe ratios of 20:80; 50:50; 80:20. The ratio if 80:20 resulted in thebest separation of CoQ10 and CoQ10H2 peaks.

Example 7—Standard Curves

TABLE 6 Preparation of samples for standard curve Conc Label Stock UsedVol of stock Diluent Vol Diluent (ng/mL) L8 1:1   1 mL Hexane 0 2500OXD:RED L7 L8 500 μL Hexane 500 μL 1250 L6 L7 500 μL Hexane 500 μL 625L5 L6 500 μL Hexane 500 μL 323 L4 L5 500 μL Hexane 500 μL 157 L3 L4 500μL Hexane 500 μL 79 L2 L3 500 μL Hexane 500 μL 39 L1 L2 500 μL Hexane500 μL 19

The samples were separated by liquid chromatography using methodsdescribed in the Example 6 with mobile phase A:B 80:20. Shown in FIG. 4are standard curves obtained for CoQ10 and CoQ10H₂.

When samples for the standard curve were prepared using HPLC grade wateras a diluent instead of hexane, with the same LC-MS/MS conditions, andwere used for the run, no detectable area counts were seen at any of theconcentrations for the reduced and the oxidized forms of CoQ₁₀.

The above experiment was repeated using different extraction solvents inan attempt to extract CoQ₁₀ from water. The extraction solvents testedwere 100% isopropyl alcohol, 100% Hexane, and 20:80 Ethanol:Hexane.Three sets of samples for standard curves were extracted using eachextraction solvent. It was observed that the area recoveries within allsamples extracted with the same extraction solvent were similar, but thearea recoveries differed for each extraction solvent group None of theextraction solvents gave a linear relationship between area counts andspiked concentration. It was ascertained that HPLC grade water did notuniformly homogenize with the standards in hexane, probably due towater's high polarity.

To tackle the problem of immiscibility of hexane and water, the CoQ10and CoQ10H₂ stocks in hexane were evaporated under a gentle stream ofnitrogen and reconstituted in methanol. These reconstituted standardswere then used to prepare samples for the standard curve with HPLC wateras a diluent, and the standards were analyzed using the above LC-MS/MSprocedure. These samples gave a linear curve for the area responses whenplotted against concentration, as is shown in FIG. 6.

CoQ10-d6 internal standard was also reduced using the same procedure asdescribed above. When a serial dilution of the internal standards weremade, with hexane evaporated and reconstituted in methanol, similarlinear relationship was observed.

CoQ10 and CoQ10H₂ stocks in hexane were evaporated and reconstituted inmethanol. CoQ10-d6 and CoQ10H₂-d6 stocks in hexane were also evaporatedand reconstituted in methanol. These reconstituted standards were thenused to prepare samples for the standard curve with HPLC water as adiluent. 100 μL of each sample was aliquoted into new tubes andextracted using three different extraction solvents described above. Thearea responses for all concentrations within the same extraction solventgroup were found to be similar, with no linear relationship between arearesponse and concentrations fortified.

CoQ10, CoQ10H₂, CoQ10-d6 and CoQ10H₂-d6 stocks in hexane were evaporatedand reconstituted in methanol. The reconstituted standards were thenused to prepare samples for the standard curve with plasma as a diluentto give a linear concentration range of 19-2500 ng/ml of CoQ10 andCoQ10H₂. 20 μL of 25 μg/mL of CoQ10-d6 and CoQ10H₂-d6 s was added toeach tube. Subsequently, each sample was extracted using 1 mL ofethanol:hexane 20:80. Each sample was vortexed vigorously for 4-5 min,centrifuged and 150 μL of the top layer was removed and put it intoLC-MS/MS vials for analysis. All of the above steps were carried out asquickly as possible using a cryo-block cooled to −20° C., and 10 μL fromeach tube was injected for LC-MS/MS analysis.

A linear relationship was observed between the area ratios andconcentrations spiked. Due to the high concentrations of the oxidizedand reduced forms, there was overlap between the peaks and hence nobaseline separation was observed.

In the next experiment, 50 μL of the standards was used as the startingpoint and 1.5 mL of the extracting solvent was used. Different injectionvolumes were tried. Injecting 3 μL of the standards gave a baselineseparated chromatogram with linear response and r² values of ≥0.99.

Example 8—Quantification of Total CoQ10 in Plasma Samples

In this experiment, the amount of total CoQ10 in different plasmasamples was measured. A single isopropanol extraction of each plasmasample was used to extract CoQ10, which was subsequently quantifiedusing LC-MS/MS. The standard curve, shown in the graph in FIG. 6, wasconstructed using the samples containing 0, 3, 5, 10, 50, 100, 300, 500,800 and 1000 μg/mL of CoQ10, analyzed in duplicates. The calculated %accuracy for the measurements of CoQ10 concentration in the samples usedfor the standard curve ranged from 91.3% to 113% for samples with CoQ10concentration ranging between 5 and 800 μg/mL. The results of theanalyses of plasma samples, each containing unknown amount of CoQ10 ispresented in the table 7 below.

TABLE 7 CoQ10 in plasma samples Calculated Analyte IS Peak Concen- PeakArea Area Area tration Sample Name (counts) Ratio (count) (μg/mL)Solvent 1.11 × 10² 4.54 24.4 N/A (isopropanol) Blank plasma 64.1 29.122.1 N/A Blank plasma 0  0 73.2 N/A Unknown Sample 1 7.79 × 10³ 2.62 ×10⁻² 2.97 × 10⁵ 3.07 Unknown Sample 2 6.95 × 10⁵ 2.16 3.21 × 10⁵ 299Unknown Sample 3 1.13 × 10⁶ 3.75 3.00 × 10⁵ 520 Unknown Sample 4 2.19 ×10⁶ 7.31 3.00 × 10⁵ 1010 Unknown Sample 5 3.19 × 10⁶ 11.4 2.97 × 10⁵1580

Example 9—Quantification of Total CoQ10 in Cell Supernatants

This experiment was designed to assess the uptake of CoQ10 by culturedcells after they have been exposed to formulations containing CoQ10.Caco-2 cells were cultured according to known protocols and were exposedto CoQ10 containing formulations for 0, 1, 2, 3, 4 and 6 hours. After awash and lysis, the cell lysate was extracted with isopropanol, and theamount of CoQ10 in the extract was measured by LC/MS. The standardcurve, shown in the graph in FIG. 7, was constructed using the samplescontaining 0, 0.01, 0.05, 0.1, 0.5, 1, 3, 5, 7 and 10 μg/mL of CoQ10.The calculated % accuracy for the measurements of CoQ10 concentration inthe samples used for the standard curve ranged from 87.6% to 107% forsamples with CoQ10 concentration ranging between 0.5 and 10 μg/mL. Theresults of the analyses of spiked cell culture media (Unknown SamplesA-H) and cell supernatants (unknown Samples I-L) are presented in thetable below.

TABLE 8 CoQ10 in cultured cell lysates Calculated Analyte IS PeakConcen- Peak Area Area Area tration Sample Name (counts) Ratio (count)(μg/mL) Solvent 15.3 44.4 0.346 N/A (isopropanol) Unknown Sample A 2.84× 10⁵ 33.5 8.47 × 10³ 3.93 Unknown Sample B 1.43 × 10⁵ 16.5 8.62 × 10³2.11 Unknown Sample C 7.32 × 10⁴ 85.3 8.58 × 10³ 1.14 Unknown Sample D3.30 × 10⁵ 43.4 7.62 × 10³ 4.88 Unknown Sample E 1.67 × 10⁵ 19.3 8.66 ×10³ 2.43 Unknown Sample F 7.89 × 10⁴ 9.7 8.13 × 10³ 1.29 Unknown SampleG 4.22 × 10⁴ 4.93 8.57 × 10³ 0.679 Unknown Sample H 0  0 8.35 × 10³ NoPeak Unknown Sample I 8.27 × 10⁵ 131 6.29 × 10³ 11.5 Unknown Sample J1.23 × 10⁴ 1.28 9.56 × 10³ 0.187 Unknown Sample K 8.11 × 10⁵ 131 6.21 ×10³ 11.5 Unknown Sample L 1.82 × 10⁴ 2.07 8.80 × 10³ 0.295

Example 10—Quantification of Total CoQ10 in Nasal Wash

This experiment was designed to assess the uptake of CoQ10 by the mucousmembranes in the nasal cavity after administration of the proprietarynasal formulation of CoQ10. After intranasal administration of theproprietary formulation to rats, the mucus and liquid from rats' nasalcavity was collected after 30 minutes and 1 hour, extracted withisopropanol, and the amount of CoQ10 in the extract was measured byLC-MS/MS. The standard curve, shown in the graph in FIG. 8, wasconstructed using the samples containing 0, 1, 5, 10, 50, 100, 300, 800and 1000 μg/mL of CoQ10. The calculated % accuracy for the measurementsof CoQ10 concentration in the samples used for the standard curve rangedfrom 90.5% to 114% for all standard samples. The results of the analysesof cell extracts is presented in the table below, where M1, M2, M3, M4and M5 refer to different individual rats.

TABLE 9 CoQ10 in nasal wash Time of Exposure IS Peak Calculated to NasalAnalyte Peak Area Area Concentration Sample Name Formulation Area(counts) Ratio (count) (μg/mL) Solvent N/A 2.07 × 10² 12.3 1.68 × 10¹N/A (isopropanol) M1 30 minutes 1.02 × 10³ 4.85 × 10⁻² 2.10 × 10⁴ 2.22M1 1 hour 2.19 × 10³ 1.05 × 10⁻¹ 2.09 × 10⁴ 4.15 M2 30 minutes 1.57 ×10³ 7.66 × 10⁻² 2.05 × 10⁴ 3.18 M2 1 hour 2.34 × 10³ 1.20 × 10⁻¹ 1.95 ×10⁴ 4.68 M3 30 minutes 1.70 × 10³ 9.09 × 10⁻² 1.87 × 10⁴ 3.68 M3 1 hour1.09 × 10³ 5.68 × 10⁻² 1.92 × 10⁴ 2.5 M4 30 minutes 5.21 × 10³ 2.83 ×10⁻¹ 1.84 × 10⁴ 10.3 M4 1 hour 1.04 × 10³ 5.15 × 10⁻² 2.02 × 10⁴ 2.32 M530 minutes 3.16 × 10³ 1.60 × 10⁻¹ 1.97 × 10⁴ 6.06

Example 11—Quantification of Total CoQ10 in Tissues

This experiment was designed to determine the amount of CoQ10 present indifferent tissues of mice that have been administered a proprietaryCoQ10 formulation. After administration, the mice were sacrificed after0.5, 1, 3, 8, 24 or 48 hours. The lungs, liver and kidneys werehomogenized, and the homogenate was extracted with isopropanol. Theamount of CoQ10 in the extract was measured by LC-MS/MS. The standardcurve, shown in the graph in FIG. 9, was constructed using the samplescontaining 1, 5, 10, 50, 100, 300, and 600 μg/mL of CoQ10. Thecalculated % accuracy for the measurements of CoQ10 concentration in thesamples used for the standard curve ranged from 90.2% to 121% for allstandard samples. The results of the analyses of tissue extracts ispresented in the table below, where P1 and P2 refer to two individualmice.

TABLE 10 CoQ10 in tissues Time of Exposure to IS Peak CalculatedFormulation Analyte Peak Area Area Concentration Sample Name (hours)Area (counts) Ratio (count) (μg/mL) Solvent N/A 0 0    1.2 × 10¹ N/A(isopropanol) P1 0.5 1.08 × 10⁵ 5.86 × 10⁻¹ 1.85 × 10⁵ 22.8 P1 1 1.00 ×10⁵ 5.90 × 10⁻¹ 1.70 × 10⁵ 22.9 P1 3 6.21 × 10⁴ 3.41 × 10⁻¹ 1.82 × 10⁵13.5 P1 24 5.55 × 10⁴ 3.05 × 10⁻¹ 1.82 × 10⁵ 12.2 P1 48 7.23 × 10⁴ 5.03× 10⁻¹ 1.44 × 10⁵ 19.6 P2 0.5 1.00 × 10⁵ 6.74 × 10⁻¹ 1.49 × 10⁵ 26.1 P21 8.27 × 10⁵ 6.66 1.24 × 10⁵ 252 P2 3 6.68 × 10⁴ 4.79 × 10⁻¹ 1.39 × 10⁵18.8 P2 8 6.82 × 10⁴ 3.85 × 10⁻¹ 1.77 × 10⁵ 15.2 P2 24 7.63 × 10⁴ 4.08 ×10⁻¹ 1.87 × 10⁵ 16.1 P2 48 7.10 × 10⁴ 3.63 × 10⁻¹ 1.96 × 10⁵ 14.4

Example 12—Quantification of Oxidized and Reduced Forms of CoQ10 inSerum

In this experiment, the amounts of oxidized and reduced forms of CoQ10in serum samples were measured. The samples were spiked with CoQ10-d6and CoQ10H₂-d6 internal standards, and CoQ10 and CoQ10H₂ were extractedfrom each serum sample using a single extraction with 1-propanol, andboth CoQ10 and CoQ10H₂ were quantified using LC-MS/MS. Chromatographicseparation was accomplished using the column Agilent C18 poroshell (2.1μparticle size) and Methanol:1-Propanol 80:20 with 5 mM Ammonium formateas a mobile phase. The mass spectrometric method involved simultaneousquantification of the reduced and oxidized forms of CoQ10 in a singlerun, accomplished by monitoring the transition of m/z 880 to m/z 197 forthe oxidized form of CoQ10, and the transition of m/z 882 to m/z 197 forthe reduced form of CoQ10. All samples were handled by using cooledcryoblocks to minimize the oxidation of CoQ10H₂. The standard curve wasconstructed using the samples containing 0, 20, 40, 158, 313, 625, 1250and 2500 ng/mL of CoQ10 (samples L1, L2, L3, L4, L5, L6, L7 and L8).Also analyzed were standards from the National Institute of Standardsand Technology (NIST1, NIST2 and NIST3). The calculated % accuracy forthe measurements of CoQ10 concentration in the samples used for thestandard curve ranged from 86.5% to 118% for all samples. The standardcurves for the oxidized CoQ10 and reduced CoQ10 (CoQ10H₂) and therepresentative chromatograms are shown in FIGS. 10 and 11, respectively,and the raw mass spectrometric data is presented in Table 11 below. Alldata corresponding to the samples labeled with “ox” are for the oxidizedform of CoQ10, and the data corresponding to the samples labeled with“red” correspond to the reduced form of CoQ10. The samples labeled QC1,QC2 and QC3 correspond to quality control samples comprising serumspiked with CoQ10 from a lot that is different from the one used toprepare the samples for the standard curve.

TABLE 11 Raw data for quantification of oxidized and reduced forms ofCoQ10 in serum. Analyte Analyte Analyte IS IS Calculated Sample SamplePeak Area Peak Height Concentration Peak Area Peak Height ConcentrationAccuracy Name Type (counts) (cps) (ng/mL) (counts) (cps) (ng/mL) (%)Solvent Solvent 0 0 0 0 0 N/A N/A Solvent Solvent 8.53 × 10³ 7.08 × 10²0 0 0 N/A N/A Solvent Solvent 0 0 0 0 0 N/A N/A Solvent Solvent 0 0 0 00 N/A N/A Solvent Solvent 0 0 0 0 0 N/A N/A Solvent Solvent 0 0 0 0 0N/A N/A L1-ox Standard 4.37 × 10⁴ 4.10 × 10³ 20 1.02 × 10⁶ 9.52 × 10⁴19.1 95.3 L1-red Standard 4.34 × 10³ 5.71 × 10² 20 2.29 × 10⁵ 2.79 × 10⁴23.6 118 L2-ox Standard 4.50 × 10⁴ 4.22 × 10³ 40 9.10 × 10⁵ 8.32 × 10⁴38.4 96.1 L2-red Standard 6.28 × 10³ 7.67 × 10² 40 2.10 × 10⁵ 2.56 × 10⁴40.8 102 L3-ox Unknown 5.23 × 10⁴ 5.09 × 10³ N/A 9.73 × 10⁵ 9.08 × 10⁴51.4 N/A L3-red Unknown 1.01 × 10⁴ 1.21 × 10³ N/A 2.16 × 10⁵ 2.61 × 10⁴67.1 N/A L4-ox Standard 8.10 × 10⁴ 7.70 × 10³ 158 8.80 × 10⁵ 8.17 × 10⁴165 104 L4-red Standard 1.92 × 10⁴ 2.36 × 10³ 158 2.11 × 10⁵ 2.56 × 10⁴137 86.5 L5-ox Standard 1.23 × 10⁵ 1.18 × 10⁴ 313 8.48 × 10⁵ 7.77 × 10⁴321 103 L5-red Standard 3.50 × 10⁴ 4.14 × 10³ 313 1.91 × 10⁵ 2.30 × 10⁴281 89.9 L6-ox Standard 2.08 × 10⁵ 1.94 × 10⁴ 625 8.17 × 10⁵ 7.53 × 10⁴649 104 L6-red Standard 7.08 × 10⁴ 8.25 × 10³ 625 1.78 × 10⁵ 2.10 × 10⁴619 99 L7-ox Standard 3.77 × 10⁵ 3.52 × 10⁴ 1250 8.36 × 10⁵ 7.85 × 10⁴1230 98.4 L7-red Standard 1.58 × 10⁵ 1.92 × 10⁴ 1250 1.90 × 10⁵ 2.27 ×10⁴ 1310 105 L8-ox Standard 7.57 × 10⁵ 6.99 × 10⁴ 2500 8.68 × 10⁵ 8.01 ×10⁴ 2480 99/3 L8-red Standard 3.16 × 10⁵ 3.82 × 10⁴ 2500 1.98 × 10⁵ 2.40× 10⁴ 2500 99.9 Solvent Solvent 0 0 0 0 0 N/A N/A Solvent Solvent 0 0 00 0 N/A N/A QC1-ox Quality 5.75 × 10⁴ 5.39 × 10³ 63 8.52 × 10⁵ 8.00 ×10⁴ 92.3 147 control QC1-red Quality 8.85 × 10³ 1.10 × 10³ 63 1.83 × 10⁵2.21 × 10⁴ 69.8 111 control QC2-ox Quality 1.17 × 10⁵ 1.20 × 10⁴ 2508.75 × 10⁵ 8.05 × 10⁴ 288 115 control QC2-red Quality 3.52 × 10⁴ 4.21 ×10³ 250 1.98 × 10⁵ 2.37 × 10⁴ 274 110 control QC3-ox Quality 3.76 × 10⁵3.44 × 10⁴ 1250 7.80 × 10⁵ 7.20 × 10⁴ 1330 106 control QC3-red Quality1.89 × 10⁵ 2.26 × 10⁴ 1250 2.33 × 10⁵ 2.75 × 10⁴ 1270 102 controlSolvent Solvent 0 0 0 0 0 N/A N/A Solvent Solvent 0 0 0 0 0 N/A N/ANIST1-ox Unknown 3.19 × 10⁵ 3.01 × 10⁴ N/A 8.49 × 10⁵ 8.05 × 10⁴ 1010N/A NIST1-red Unknown 1.97 × 10⁴ 1.88 × 10³ N/A 1.90 × 10⁵ 2.30 × 10⁴157 N/A NIST2-ox Unknown 3.43 × 10⁵ 3.23 × 10⁴ N/A 8.37 × 10⁵ 7.82 × 10⁴1110 N/A NIST2-red Unknown 2.22 × 10⁴ 2.10 × 10³ N/A 2.05 × 10⁵ 2.45 ×10⁴ 165 N/A NIST3-ox Unknown 4.79 × 10⁵ 4.50 × 10⁴ N/A 8.07 × 10⁵ 7.54 ×10⁴ 1650 N/A NIST3-red Unknown 3.11 × 10⁴ 2.92 × 10³ N/A 2.10 × 10⁵ 2.57× 10⁴ 227 N/A Solvent Solvent 0 0 0 0 0 N/A N/A Solvent Solvent 0 0 0 00 N/A N/A

Example 13—Quantification of Oxidized and Reduced Forms of CoQ10 inPlasma

In this experiment, the amounts of oxidized and reduced forms of CoQ10in plasma samples were measured. The samples were spiked with CoQ10-d6and CoQ10H₂-d6 internal standards, and CoQ10 and CoQ10H₂ were extractedfrom each plasma sample using extraction with hexane and 1-propanol, andboth CoQ10 and CoQ10H₂ were quantified using LC-MS/MS. Chromatographicseparation was accomplished using the column Agilent C18 poroshell (2.1μparticle size) and Methanol:1-Propanol 80:20 with 5 mM Ammonium formateas a mobile phase. The mass spectrometric method involved simultaneousquantification of the reduced and oxidized forms of CoQ10 in a singlerun, accomplished by monitoring the transition of m/z 880 to m/z 197 forthe oxidized form of CoQ10, and the transition of m/z 882 to m/z 197 forthe reduced form of CoQ10. All samples were handled by using cooledcryoblocks to minimize the oxidation of CoQ10H₂. The standard curve wasconstructed using the samples containing 0, 20, 158, 625, 1250 and 2500ng/mL of CoQ10 (samples L1, L2, L3, L4, L5, L6, L7 and L8). Thecalculated % accuracy for the measurements of CoQ10 concentration in thesamples used for the standard curve ranged from 84.8% to 119% for allsamples. The standard curves and the representative chromatograms forthe oxidized CoQ10 and reduced CoQ10 (CoQ10H₂) are shown in FIGS. 12 and13, respectively, and the raw mass spectrometric data is presented inTable 12 below. All data corresponding to the samples labeled with “ox”are for the oxidized form of CoQ10, and the data corresponding to thesamples labeled with “red” correspond to the reduced form of CoQ10. Thesamples labeled QC1, QC2, QC3 and QC4 correspond to quality controlsamples comprising plasma spiked with CoQ10 from a lot that is differentfrom the one used to prepare the samples for the standard curve.

TABLE 12 Raw data for quantification of oxidized and reduced forms ofCoQ10 in plasma. Analyte Analyte Analyte IS IS Calculated Sample SamplePeak Area Peak Height Concentration Peak Area Peak Height ConcentrationAccuracy Name Type (counts) (cps) (ng/mL) (counts) (cps) (ng/mL) (%)Solvent Solvent 0 0 0 0 0 N/A N/A Solvent Solvent 0 0 0 0 0 N/A N/AL1-ox Standard 1.88 × 10⁵ 1.72 × 10³ 20 9.38 × 10⁵ 8.32 × 10⁴ 23.8 119L1-red Standard 6.77 × 10⁴ 7.88 × 10⁴ 20 3.36 × 10⁵ 3.92 × 10⁴ 19.6 98.1L2-ox Standard 1.77 × 10⁵ 1.61 × 10³ N/A 8.80 × 10⁵ 7.82 × 10⁴ 26.1 N/AL2-red Standard 6.94 × 10⁴ 8.06 × 10⁴ N/A 3.13 × 10⁵ 3.68 × 10⁴ 46.6 N/AL3-ox Unknown 1.65 × 10⁵ 1.49 × 10⁴ N/A 6.75 × 10⁵ 5.94 × 10⁴ 130 N/AL3-red Unknown 9.19 × 10⁴ 1.09 × 10⁴ N/A 3.50 × 10⁵ 4.15 × 10⁴ 101 N/AL4-ox Standard 1.93 × 10⁵ 1.77 × 10⁴ 158 7.87 × 10⁵ 7.02 × 10⁴ 134 84.8L4-red Standard 8.64 × 10⁴ 1.04 × 10⁴ 158 2.79 × 10⁵ 3.36 × 10⁴ 165 104L5-ox Standard 2.44 × 10⁵ 2.25 × 10⁴ N/A 8.54 × 10⁵ 7.70 × 10⁴ 230 N/AL5-red Standard 1.14 × 10⁵ 1.35 × 10⁴ N/A 2.99 × 10⁵ 3.57 × 10⁴ 260 N/AL6-ox Standard 3.57 × 10⁵ 3.24 × 10⁴ 625 8.18 × 10⁵ 7.43 × 10⁴ 598 95.7L6-red Standard 1.83 × 10⁵ 2.25 × 10⁴ 625 2.88 × 10⁵ 3.47 × 10⁴ 601 96.1L7-ox Standard 5.73 × 10⁵ 5.32 × 10⁴ 1250 8.30 × 10⁵ 7.63 × 10⁴ 1210 97L7-red Standard 3.20 × 10⁵ 3.91 × 10⁴ 1250 2.82 × 10⁵ 3.33 × 10⁴ 1270101 L8-ox Standard 9.97 × 10⁵ 9.38 × 10⁴ 2500 7.94 × 10⁵ 7.14 × 10⁴ 2580103 L8-red Standard 5.95 × 10⁵ 7.08 × 10⁴ 2500 2.90 × 10⁵ 3.48 × 10⁴2500 100 Solvent Solvent 0 0 0 0 0 N/A N/A Solvent Solvent 0 0 0 0 0 N/AN/A Solvent Solvent 0 0 0 0 0 N/A N/A Solvent Solvent 0 0 0 0 0 N/A N/AQC1-ox Quality 2.04 × 10⁵ 1.83 × 10⁴ 125 7.92 × 10⁵ 7.21 × 10⁴ 164 131control QC1-red Quality 9.85 × 10⁴ 1.15 × 10⁴ 125 3.23 × 10⁵ 3.83 × 10⁴158 126 control QC2-ox Quality 2.21 × 10⁵ 1.98 × 10⁴ 250 7.20 × 10⁵ 6.49× 10⁴ 283 113 control QC2-red Quality 1.40 × 10⁵ 1.66 × 10⁴ 250 3.65 ×10⁵ 4.30 × 10⁴ 263 105 control QC3-ox Quality 5.61 × 10⁵ 5.06 × 10⁴ 12507.82 × 10⁵ 7.19 × 10⁴ 1280 102 control QC3-red Quality 3.86 × 10⁵ 4.65 ×10⁴ 1250 3.93 × 10⁵ 4.83 × 10⁴ 1070 85.3 control QC4-ox Quality 1.15 ×10⁶ 1.04 × 10⁵ 2500 9.09 × 10⁵ 8.18 × 10⁴ 2610 104 control QC4-redQuality 6.16 × 10⁵ 7.51 × 10⁴ 2500 3.13 × 10⁵ 3.69 × 10⁴ 2380 95.3control

IV. Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

V. Related References

All publications and patent documents cited in this application areincorporated by reference in pertinent part for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their disclosure. It is to be understood that while the presentdisclosure has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the present disclosure, which is defined bythe scope of the appended claims. Other aspects, advantages, andmodifications are within the scope of the following claims and theirequivalents.

All figures are offered by way of illustration, not by way oflimitation. While specific examples have been provided, the descriptionsare illustrative and not restrictive. Any one or more of the features ofthe previously described embodiments can be combined in any manner withone or more features of any other embodiments in the present disclosure.Furthermore, many variations of the present disclosure will becomeapparent to those skilled in the art upon review of this disclosure.

What is claimed is:
 1. An isotopically labeled analog of the oxidizedform of coenzyme Q10 CoQ10-d6 of the following formula:


2. An isotopically labeled analog of the reduced form of coenzyme Q10CoQ10H₂-d6 of the following formula:


3. A mixture for analysis by LC/MS/MS comprising a biological sample andthe isotopically labeled analog of claim
 1. 4. The mixture of claim 3,wherein said biological sample is a bodily fluid.
 5. The mixture ofclaim 4, wherein said bodily fluid is blood.
 6. The mixture of claim 3,wherein said biological sample is serum.
 7. The mixture of claim 3,wherein said biological sample is plasma.
 8. The mixture of claim 3,wherein said biological sample is a human sample.
 9. A mixture foranalysis by LC/MS/MS comprising a biological sample and the isotopicallylabeled analog of claim
 2. 10. The mixture of claim 9, wherein saidbiological sample is a bodily fluid.
 11. The mixture of claim 10,wherein said bodily fluid is blood.
 12. The mixture of claim 9, whereinsaid biological sample is serum.
 13. The mixture of claim 9, whereinsaid biological sample is plasma.
 14. The mixture of claim 9, whereinsaid biological sample is a human sample.